Tlr3 binding agents

ABSTRACT

The present invention relates to antibodies (e.g. monoclonal antibodies), antibody fragments, and derivatives thereof that specifically bind TLR3, and that optionally further modulate, e.g. inhibit, signaling. The invention also relates to cells producing such antibodies; methods of making such antibodies; fragments, variants, and derivatives of the antibodies; pharmaceutical compositions comprising the same; methods of using the antibodies to diagnose, treat or prevent diseases, e.g. autoimmune diseases, inflammatory diseases and the like.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 13/102,019 filed May 5, 2011, entitled “TLR3 Binding Agents”,which claims priority to and is a continuation of PCT InternationalApplication Number PCT/EP2010/059946, filed on Jul. 9, 2010, entitled“TLR3 Binding Agents”, and published in English on Jan. 13, 2011, whichclaims priority to U.S. patent application 61/224,548, filed on Jul. 10,2009. The disclosures of each of the above-described applications arehereby incorporated herein by reference in their entireties.

FIELD OF THE INVENTION

The present invention relates to antibodies (e.g. monoclonalantibodies), antibody fragments, and derivatives thereof thatspecifically bind TLR3, and that optionally further modulate, e.g.inhibit, signaling. The invention also relates to cells producing suchantibodies; methods of making such antibodies; fragments, variants, andderivatives of the antibodies; pharmaceutical compositions comprisingthe same; methods of using the antibodies to diagnose, treat or preventdiseases, e.g. autoimmune diseases, inflammatory diseases and the like.

BACKGROUND

Drosophila toll proteins control dorsal-ventral patterning and arethought to represent an ancient host defense mechanism. In humans, TLRsare believed to be an important component of innate immunity Human andDrosophila Toll protein sequences show homology over the entire lengthof the protein chains. The family of human Toll-like receptors iscomprised of ten highly conserved receptor proteins, TLR1-TLR10. LikeDrosophila toll, human TLRs are type I transmembrane proteins with anextracellular domain consisting of a leucine-rich repeat (LRR) domainthat recognizes pathogen-associated molecular patterns (PAMPs), and acytoplasmic domain that is homologous to the cytoplasmic domain of thehuman interleukin-1 (IL-1) receptor. Similar to the signaling pathwaysfor both Drosophila toll and the IL-1 receptor, human Toll-likereceptors signal through the NF-κB pathway.

Although the different mammalian TLRs share many characteristics andsignal transduction mechanisms, their biological functions are verydifferent. This is due in part to the fact that four different adaptormolecules (MyD88, TIRAP, TRIF and TRAF) are associated in variouscombinations with the TLRs and mediate different signaling pathways. Inaddition, different ligands for one TLR may preferentially activatedifferent signal transduction pathways. Furthermore, the TLRs aredifferentially expressed in various hematopoietic and non-hematopoieticcells. Accordingly, the response to a TLR ligand depends not only on thesignal pathway activated by the TLR, but also on the nature of the cellsin which the individual TLR is expressed.

Toll-like receptor 3 (TLR3) has received considerable attention as atherapeutic target as TLR3 signaling has been implicated in inflammatoryand autoimmune conditions. Patent application WO98/50547 provides thenucleic acid and amino acid sequence of the hTLR3 protein. LeBouteilleret al. (2005) J. Biol. Chem. 280(46): 38133-38145) disclose use of ananti-TLR3 antibody to bind cell surface TLR3. Antibody C1130 is statedto be activatory toward TLR3 and has been described in WO 2007/051164.Polyclonal antibodies that inhibited TLR3 were described in Cavassani etal. (2008) J. Exp. Med. 205: 2609-2621, WO 03/106499 and Matsumoto etal. (2003) J. Immunol. 171:3154-3162 describes an antibody correspondingto antibody clone TLR3.7 (eBioScience Inc., San Diego) reported to bindand inhibit cell surface TLR3 but not cell compartment TLR3 or inmyeloid-lineage DC. WO 06/060513 describes an antibody C1068 which isreported to inhibit cytokine production in epithelial cells, which arereported to express TLR3 on the cell surface. C1068 is stated to competewith antibody TLR3.7 for binding to TLR3 (see WO2010/051470). PCT patentapplication WO2010/051470 provides anti-TLR3 antibodies. Such antibodiesare stated to block dsRNA and are proposed to prevent binding of dsRNAto TLR3. Other anti-TLR3 antibodies for research use include polyclonalanti-TLR3 antibodies from R&D Systems Corp., antibody 40C1285 from Abcamand antibodies 619F7, 713E4, 716G10, IMG-5631 and -IMG-5348, all fromImgenex Corp.

However, while several anti-TLR3 antibodies have been generated to date,these antibodies have generally been intended for research only, and notfor therapeutic use. As further described herein, the present disclosureshows that among currently available anti-TLR3 antibodies, while theymay be useful in some research settings to make experimentalobservations, they are not optimally suited for use as therapeuticagents, e.g. to modulate TLR3. There is therefore a need to provideimproved antibodies directed to TLR3.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides novel compositionscomprising, and methods of using monoclonal antibodies, including butnot limited to antibody fragments, and derivatives that specificallybind human TLR3. In one aspect, the antibodies inhibit TLR3 signalingwithout blocking the binding of a TLR3 ligand to a TLR3 polypeptide. Inone aspect, the antibodies bind human TLR3 under acidic conditions, andin particular under conditions representative of that encountered in anacidified subcellular compartment of a cell (e.g. compartments of theendocytic pathway endosomic, lysosomal). Such acidic conditions aregenerally characterized by a pH lower than about pH 6.5, or betweenabout pH 4.5 to 6.5, or about pH 5.6.

In one aspect of any of the embodiments herein, the antibodies modulate,optionally inhibit, TLR3 signaling in an acidified subcellularcompartment of a cell (e.g. compartments of the endocytic pathwayendosomic, lysosomal).

In one aspect of any of the embodiments herein, the antibodies modulate,optionally inhibit, TLR3 signaling in a dendritic cell (DC) (e.g. amyeloid DC, monocyte derived DC).

In one aspect of any of the embodiments herein, the antibodies canoptionally be characterized as not having substantially lower affinityfor binding human TLR3 under acidic conditions than under neutralconditions e.g. where the K_(D) for binding to TLR3 decreases by no morethan 0.2-, 0.3-, −0.4, 0.5-, 1.0-, or 1.5-log₁₀. Neutral conditions aregenerally characterized by a pH between 6.6 and 7.4, for example aslightly alkaline pH of 7.2 found in the cell cytosol. Optionally, theantibodies do not have substantially different (lower or higher)affinity for binding human TLR3 under acidic conditions than underneutral conditions e.g. where the K_(D) for binding to TLR3 underneutral and acidic conditional differs by no more than 0.2-, 0.3-, 0.4-,0.5-, 1.0-, or 1.5-log₁₀.

In other aspects of any of the embodiments herein, the antibodies'bivalent binding affinity for TLR3 under acidic conditions canoptionally be characterized by a mean K_(D) of no more than about (i.e.better affinity than) 100, 50, 10, 5, or 1 nanomolar, preferablysub-nanomolar or optionally no more than about 300, 200, 100 or 10picomolar.

In other aspects of any of the embodiments herein, the antibodiesinhibit TLR3 signaling without blocking the binding of a TLR3 ligand toa TLR3 polypeptide. The TLR3 ligand will generally be a ligand otherthan an anti-TLR3 antibody and may be a naturally occurring ornon-naturally occurring TLR3 ligand, optionally a dsRNA-based ligandsuch as polyAU (polyadenylic acid:polyuridylic acid) or polyIC(polyinosinic:polycytidylic acid). In particular, the inventors haveestablished that the antibodies according to the invention are able toinhibit TLR3 signaling even when a TLR3 ligand such as dsRNA is alreadybound to the TLR3 polypeptide. The antibodies according to the inventionare also able to inhibit TLR3 signaling even in a pre-actived condition,e.g., in the presence of IFNα. The antibodies according to the inventionare believed to be effective to treat a patient having an establishedautoimmune disease, e.g. naturally occurring TLR3 ligand such as dsRNAand/or the presence of, and in particular, high levels of, IFNα in thediseased cells. The antibodies will also have the advantage of bindingTLR3 even if the TLR3 ligand binding site is occupied by a dsRNAmolecule thus potentially allowing broader overall binding.

The present disclosure shows that the antibodies that bind human TLR3under acidic conditions have a strong ability to modulate, particularlyinhibit, TLR3 signaling in cells (myeloid dendritic cells (MdDC);monocyte derived DC (MoDC)) which express TLR3 solely or primarily intheir cytoplasmic compartments, and primarily in compartments of theendocytic pathway (e.g. endosomes). The antibodies bind to a region inTLR3 which is not involved in binding to dsRNA, and the antibodies donot prevent dsRNA from binding to TLR3 under acidic conditions. Thecompositions and methods are useful for a multitude of applications, andare particularly well suited to modulating TLR3 signaling (e.g. in vivo)where cytosolic (e.g. endocytic pathway compartment-localized) TLR3 istargeted. Modulating cytosolic TLR3 signaling can be useful to treat orprevent a disease for which modulation of TLR3 signaling in DC or othercells that express TLR3 in acidic cytosolic compartments (e.g. inendosomes) is beneficial. For example, inhibiting TLR3 signaling in DC(e.g. as observed by inhibition of cytokine production by the DC) can beused the treatment or prevention of inflammatory or autoimmune disorderssince DC have a well documented capacity to take up antigens fromapoptotic or necrotic cells (Albert et al (2004) Nat. Rev. Immunol. 4:223-231), including during tissue necrosis during acute inflammation(Cavassani et al. (2008). Optionally, the antibodies inhibit TLR3signaling, e.g. inhibit cytokine production (e.g. IP10) induced by thestimulation of a TLR3 receptor by a TLR3 ligand.

Endosomes and lysosomes are membrane bound compartments inside cells,form part of the endocytic pathway and are usually acidic due to theaction of a proton-pumping ATPase of the endosomal membrane. Theearliest measurements of in situ lyosomal pH found a pH of 4.7-4.8 inmacrophages; the pH of fibroblast endosomes involved inreceptor-mediated endoxytosis was determined to be about 5.5. Earlystudies of TLR3 identified it as being expressed in the cytosol inmonocyte-derived DCs and that it probably bound its ligand insubcellular compartments of the endocytic pathway (Matsumoto et al.(2003) J. Immunol. 171:3154-3162). TLR3 has since been reported to beexpressed in cells' endosomic compartment in dendritic cells,astrocytes, macrophages, T cells, epithelial cells, fibroblasts andhepatocytes, although TLR3 has also been found on cell surface,particularly on epithelial cells, and in some cases of inflammation alsoon macrophages (Cavassani et al. 2008, supra). Endosomal acidificationhas been shown to have a role in TLR3 signaling since treatment withchloroquine, an inhibitor of endosomal acidification, inhibits TLR3signaling in DC. The antibodies provided herein that bind TLR3 underacidic conditions corresponding to an acidified endosomic compartment(e.g. pH of about 5.6, or less than about 6.5) have the advantage ofallowing efficient high affinity binding to, and optionally furthermodulation of, TLR3 in endosomic compartments compared to antibodiesthat lose their affinity under acidic conditions and thus may exerttheir effects more on cell surface TLR3. The antibodies exemplified havestrong inhibitory activity on TLR3 in DC which are known for expressingTLR3 primarily in cytosomal compartments.

In one embodiment, the present invention provides monoclonal antibodiesthat specifically bind human TLR3 and inhibit TLR3 signaling, e.g.inhibit cytokine production induced by the stimulation of a TLR3receptor by a TLR3 ligand, without blocking the binding of a ligand ofTLR3 (e.g. a natural or synthetic ligand of TLR3, a nucleic acid basedligand, a dsRNA, viral dsRNA, polyIC, polyAU) to a TLR3 polypeptide.When TLR3 polypeptides are bound by such antibodies, dsRNA can stillbind the TLR3 polypeptides, reducing dsRNA available to bind toremaining non antibody-bound TLR3 and/or other dsRNA receptors (i.e.RIG-I, MDA-5, TLR7, etc), thereby potentially reducing undesirable sideeffects such as increased toxicity, inappropriate signaling cascadeactivation and so on, and resulting conditions, e.g. chronicinflammation, that arise from dsRNA induced signaling. Such antibodycompositions and methods are useful for a multitude of applications,particularly to treat or prevent a disease related to TLR3 signaling,and in view of their mechanism of action, the antibodies of theinvention can be used for anergizing or inhibiting TLR3 polypeptides.Optionally, the antibody can be characterized as not detectably reducingthe binding of a double-stranded RNA ligand of TLR3 to a TLR3polypeptide. The antibody may or may not also be capable of binding withhigh affinity to human TLR3 under acidic conditions, e.g. underconditions representative of that encountered in an acidified endosomiccompartment. In one embodiment, where an antibody is sought that caninhibit signaling by TLR3, it will be advantageous that an antibody thatspecifically binds TLR3 and inhibits TLR3 signaling without blocking thebinding of a double-stranded RNA ligand of TLR3 to a TLR3 polypeptidecan additionally be capable of binding and inhibiting human TLR3 underacidic conditions as described herein, and in particular underconditions representative of that encountered in an acidified endosomiccompartment of a cell.

In one aspect of any of the embodiments of the invention, the antibodycompetes for binding to a TLR3 polypeptide with any one or anycombination of monoclonal antibodies 31C3, 29H3, 23C8, 28F11 or 34A3,optionally under acid and/or neutral conditions. In one embodiment, anantibody of the invention competes for binding to a TLR3 polypeptide,optionally under acid and/or neutral conditions, with an antibodyselected from the group consisting of:

(a) an antibody having respectively a VH and VL region of SEQ ID NOS 2and 3 (31C3),

(b) an antibody having respectively a VH and VL region of SEQ ID NOS 10and 11 (29H3),

(c) an antibody having respectively a VH and VL region of SEQ ID NOS 18and 19 (28F11),

(d) an antibody having respectively a VH and VL region of SEQ ID NOS 26and 27 (23C8) and

(e) an antibody having respectively a VH and VL region of SEQ ID NOS 34and 35 (34A3).

In the foregoing aspect, the antibody may compete for binding to a TLR3polypeptide with an antibody selected from the group consisting of: (a)an antibody having respectively a VH and VL region of SEQ ID NOS 2 and 3(31C3), (b) an antibody having respectively a VH and VL region of SEQ IDNOS 10 and 11 (29H3), (c) an antibody having respectively a VH and VLregion of SEQ ID NOS 18 and 19 (28F11), (d) an antibody havingrespectively a VH and VL region of SEQ ID NOS 26 and 27 (23C8) and (e)an antibody having respectively a VH and VL region of SEQ ID NOS 34 and35 (34A3) under acid conditions. The antibody may be a monoclonalantibody. The antibody may be in a composition comprising apharmaceutically acceptable carrier. Some aspects relate to a method fortreating or preventing a disease wherein the inhibition of TLR3signaling pathway is desirable comprising administering atherapeutically effective amount of an antibody of the foregoing aspectsto a patient in need thereof.

The antibody may compete for binding to a TLR3 polypeptide with anantibody selected from the group consisting of: (a) an antibody havingrespectively a VH and VL region of SEQ ID NOS 2 and 3 (31C3), (b) anantibody having respectively a VH and VL region of SEQ ID NOS 10 and 11(29H3), (c) an antibody having respectively a VH and VL region of SEQ IDNOS 18 and 19 (28F11), (d) an antibody having respectively a VH and VLregion of SEQ ID NOS 26 and 27 (23C8) and (e) an antibody havingrespectively a VH and VL region of SEQ ID NOS 34 and 35 (34A3) underneutral conditions. The antibody may be a monoclonal antibody. Theantibody may be in a composition comprising a pharmaceuticallyacceptable carrier. Some aspects relate to a method for treating orpreventing a disease wherein the inhibition of TLR3 signaling pathway isdesirable comprising administering a therapeutically effective amount ofan antibody of the foregoing aspects to a patient in need thereof.

Any of the antibodies described herein may be used in a method fortreating or preventing a disease wherein the inhibition of TLR3signaling pathway is desirable comprising administering atherapeutically effective amount of the antibody to a patient in needthereof. In some aspects, the disease is selected from the groupconsisting of autoimmunity, inflammation, allergy, asthma, infection,cirrhosis, sepsis, diabetes, and viral infections.

In one embodiment the antibody competes for binding to a TLR3polypeptide with antibodies 31C3 and 29H3; in one embodiment theantibody competes for binding to a TLR3 polypeptide with antibodies 31C3and 23C8; in one embodiment the antibody competes for binding to a TLR3polypeptide with antibodies 31C3 and 28F11; in one embodiment theantibody competes for binding to a TLR3 polypeptide with antibodies 31C3and 34A3. In one embodiment the antibody competes for binding to a TLR3polypeptide with antibodies 29H3 and 23C8; in one embodiment theantibody competes for binding to a TLR3 polypeptide with antibodies 29H3and 28F11; in one embodiment the antibody competes for binding to a TLR3polypeptide with antibodies 29H3 and 34A3. In one embodiment theantibody competes for binding to a TLR3 polypeptide with antibodies 23C8and 28F11; in one embodiment the antibody competes for binding to a TLR3polypeptide with antibodies 23C8 and 34A3. In one embodiment theantibody competes for binding to a TLR3 polypeptide with antibodies28F11 and 34A3.

In one embodiment, the antibody of the invention comprises a light chaincomprising:

-   -   (a) a light chain CDR1 (LCDR1) amino acid sequence selected from        SEQ ID NOS: 61, 64 and 65;    -   (b) a light chain CDR2 (LCDR2) amino acid sequence selected from        SEQ ID NOS: 62, 66 and 67; and/or    -   (c) a light chain CDR3 (LCDR3) amino acid sequence selected from        SEQ ID NOS: 63, 68, 69 and 70.

In one embodiment, the antibody of the invention comprises a heavy chaincomprising:

-   -   (a) a heavy chain CDR1 (HCDR1) amino acid sequence selected from        SEQ ID NOS: 71 to 76;    -   (b) a heavy chain CDR2 (HCDR2) amino acid sequence selected from        SEQ ID NOS: 77 to 81; and/or    -   (c) a heavy chain CDR3 (HCDR3) amino acid sequence selected from        SEQ ID NOS: 82 to 85.

In one embodiment, the antibody of the invention is selected from thegroup consisting of:

-   -   (a) an antibody having (i) the heavy chain CDR 1, 2 and 3        (HCDR1, HCDR2, HCDR3) amino acid sequences as shown in SEQ ID        NO: 4, 5 and 6, and (ii) the light chain CDR 1, 2 and 3 (LCDR1,        LCDR2, LCDR3) amino acid sequences as shown in SEQ ID NO: 7, 8        and 9, respectively;    -   (b) an antibody having (i) the heavy chain CDR 1, 2 and 3        (HCDR1, HCDR2, HCDR3) amino acid sequences as shown in SEQ ID        NO: 12, 13 and 14, and (ii) the light chain CDR 1, 2 and 3        (LCDR1, LCDR2, LCDR3) amino acid sequences as shown in SEQ ID        NO: 15, 16 and 17, respectively;    -   (c) an antibody having (i) the heavy chain CDR 1, 2 and 3        (HCDR1, HCDR2, HCDR3) amino acid sequences as shown in SEQ ID        NO: 20, 21 and 22, and (ii) the light chain CDR 1, 2 and 3        (LCDR1, LCDR2, LCDR3) amino acid sequences as shown in SEQ ID        NO: 23, 24 and 25, respectively;    -   (d) an antibody having (i) the heavy chain CDR 1, 2 and 3        (HCDR1, HCDR2, HCDR3) amino acid sequences as shown in SEQ ID        NO: 28, 29 and 30, and (ii) the light chain CDR 1, 2 and 3        (LCDR1, LCDR2, LCDR3) amino acid sequences as shown in SEQ ID        NO: 31, 32 and 33, respectively; and    -   (e) an antibody having (i) the heavy chain CDR 1, 2 and 3        (HCDR1, HCDR2, HCDR3) amino acid sequences as shown in SEQ ID        NO: 36, 37 and 38, and (ii) the light chain CDR 1, 2 and 3        (LCDR1, LCDR2, LCDR3) amino acid sequences as shown in SEQ ID        NO: 39, 40 and 41, respectively;    -   optionally wherein one, two, three or more of the amino acids in        any of said sequences may be substituted by a different amino        acid.

In one embodiment of the invention, the antibody binds to the same TLR3epitope as any one or any combination of monoclonal antibodies 31C3,29H3, 23C8, 28F11 or 34A3. In another embodiment, the antibody comprisesan antigen binding region of antibody 31C3. In another embodiment, theantibody comprises an antigen binding region of antibody 29H3. Inanother embodiment, the antibody comprises an antigen binding region ofantibody 23C8. In another embodiment, the antibody comprises an antigenbinding region of antibody 28F11. In another embodiment, the antibodycomprises an antigen binding region of antibody 34A3. In anotherembodiment, the antibody comprises a light chain comprising one, two orall three CDRs of the 31C3, 29H3, 23C8, 28F11 or 34A3 light chainvariable region sequence, and/or a heavy chain comprising one, two orall three CDRs of the 31C3, 29H3, 23C8, 28F11 or 34A3 heavy chainvariable region sequence. In another embodiment, the antibody is 31C3,29H3, 23C8, 28F11 or 34A3 or a fragment or derivative thereof,optionally fused to a human Fc region. Antibodies 29H3.7 and 31C3.1 havebeen deposited at the Collection Nationale de Culture de Microorganismes(CNCM), Institut Pasteur, 25 rue de Docteur Roux, F-75724 Paris on 3Jul. 2009, under the number CNCM I-4187 and CNCM I-4186 respectively.Antigen binding regions of antibodies 31C3, 29H3, 23C8, 28F11 or 34A3are also disclosed in SEQ ID NOS 2 to 41.

In one aspect of the invention, the light chain of one antibodyaccording to the present invention is obtained from or encoded by anucleic acid sequence derived from a VL gene rearrangement selected fromVK 19-14, VK aq4, VK 12-41 and VK 12-44 for the V gene, and JK2 for theJ gene.

In one aspect of the invention, the heavy chain of one antibodyaccording to the present invention is obtained from or encoded by anucleic acid sequence derived from a VH gene rearrangement selected fromVH36-60.a1.85, VH L558.1 and VH J558.2 for the V gene, and JH4 or JH2for the J gene.

In another aspect of the invention, the antibody has one or more CDRs ofthe sequence selected from the group consisting of SEQ ID NOS: 4 to 9,12 to 17, 20 to 25, 28 to 33, 36 to 41, wherein one, two, three or moreof these amino acids may be substituted by a different amino acid.

In another aspect, the invention provides an antibody that specificallybinds TLR3, wherein the antibody has one or more of the followingproperties:

-   -   a. has a subnanomolar affinity for a TLR3 polypeptide at an        acidic pH, e.g. a pH less than about 6.5, or between about 4.5        to 6.5 or about pH 5.6; or    -   b. is able to inhibit TLR3 signaling in the presence of a TLR3        ligand; or    -   c. is able to inhibit TLR3 signaling in an inflammatory        background, e.g. in the presence of inflammatory cytokines such        as IFNα; or    -   d. competes for binding to a TLR3 polypeptide with 31C3, 29H3,        28F11, 23C8 or 34A3;    -   e. does not compete with dsRNA for binding to a TLR3        polypeptide.

In one embodiment, the antibody has above-listed properties (a) and (b);(a), (b) and (c); (a), (b), (c) and (d); or (a), (b), (c), (d) and (e).In one embodiment, the antibody has properties (a) and (c); (a), (c) and(d); or (a), (c), (d) and (e). In one embodiment, the antibody hasproperties (a) and (d); (a) and (e); or (a), (d) and (e). In oneembodiment, the antibody has properties (b) and (c); (b), (c) and (d);or (b), (c), (d) and (e). In one embodiment, the antibody has properties(b) and (d); (b) and (e); or (b), (d) and (e). In one embodiment, theantibody has properties (c) and (d); (c) and (e); or (c), (d) and (e).In one embodiment, the antibody has properties (d) and (e). In anotherembodiment, the antibody additionally has any of the properties ofanti-TLR3 antibodies described herein.

In another embodiment, the antibody of any of the embodiments herein iscapable of being internalized by a cell that expresses TLR3 polypeptideon its surface.

In an embodiment of any aspect of the invention, the amino acidsequences listed in the SEQ IDs comprise one, two, three or more aminoacid substitution. In another embodiment, in any embodiment of theinvention, the embodiment may encompass an amino acid sequence may haveat least 95%, 97%, 98%, or 99% identity with an amino acid sequence in aparticular SEQ ID NO.

In another embodiment, the invention provides a monoclonal anti-TLR3antibody that has the same epitopic specificity as an antibody selectedfrom the group consisting of 31C3, 29H3, 28F11, 23C8 and 34A3.

In one embodiment, the antibody is chimeric, e.g. contains a non-murine,optionally a human, constant region. In one embodiment, the antibody ishuman or humanized. In another embodiment, the antibody is a mouseantibody. In another embodiment, the antibody does not substantiallybind to other human TLRs (e.g. TLR4).

In one aspect of any of the embodiments of the invention, the isotype ofthe antibody is IgG, optionally IgG1 or IgG3. In one embodiment theantibody comprises an Fc domain or is of an isotype that is bound byFcγR.

In one aspect of any of the embodiments of the invention, the antibodyis an antibody fragment selected from Fab, Fab′, Fab′-SH, F(ab′)2, Fv,diabodies, single-chain antibody fragment, or a multispecific antibodycomprising multiple different antibody fragments. In one aspect of anyof the embodiments of the invention, the antibody does not comprise anFc domain or is of an isotype that is not substantially bound by FcγR.In one embodiment, the antibody is of an IgG4 or IgG2 isotype. Asdemonstrated in the Examples, F(ab′)2 fragments of the antibodies of thepresent invention retained their ability to modulate TLR3 signaling inDCs and were thus taken up by DC despite their lack of Fc domain. It haspreviously generally been thought that antibodies will enter theendosomal pathway in DC at least in part by Fc receptor-mediated uptake(human DC express several types of Fcγ receptors (FcγR), including typeI (FcγRI, CD64) and type II (FcγRII, CD32)). The finding that isotypesand formats that do not bind FcγR can modulate TLR3 in DC enablesantibodies to be developed that retain desired characteristics without arisk of inducing unwanted depletion (e.g. via FcγR-mediated antibodydependent cellular cytotoxicity) of TLR3-expressing cells. For exampleIgG4 isotypes or other IgG isotypes modified to reduce their FcγRbinding can be used for their advantageous pharmacological propertiessuch as serum half-life, while modulating TLR3 signaling, in e.g. a DC,without induce the death of the cell. In one aspect of any of theembodiments of the invention, the anti-TLR3 antibody inhibits TLR3signaling and comprises a constant region of IgG4 or IgG2 isotype. Inone aspect, of any of the embodiments of the invention, the anti-TLR3antibody inhibits TLR3 signaling and comprises a constant region thatdoes not substantially bind FcγR.

In another embodiment, the antibody is conjugated or covalently bound toa detectable or toxic moiety.

In another aspect, the present invention provides a cell, e.g. ahybridoma or recombinant host cell, producing an anti-TLR3 antibody ofthe invention. In one embodiment, the cell is clone 31C3, 29H3, 23C8,28F11 or 34A3. In a related aspect, the present invention provides ahybridoma comprising: a) a B cell from a non-human mammalian host thathas been immunized with an antigen that comprises the TLR3 epitopespecifically recognized by the 31C3, 29H3, 23C8, 28F11 or 34A3 antibody,fused to b) an immortalized cell, wherein the hybridoma produces amonoclonal antibody that specifically binds to the epitope. In oneembodiment of these aspects, the monoclonal antibody binds to the sameepitope as antibody 31C3, 29H3, 23C8, 28F11 or 34A3. Optionally, thecell produces an antibody having the antigen binding region of antibody31C3, 29H3, 23C8, 28F11 or 34A3.

In another aspect, the present invention provides a method of testing anantibody, the method comprising the steps of: testing an antibody todetermine whether it:

-   -   a) inhibits TLR3 signaling, optionally without blocking the        binding of a TLR3 ligand (e.g. dsRNA) to a TLR3 polypeptide,        and/or    -   b) competes for binding to a TLR3 polypeptide with antibody        31C3, 29H3, 23C8, 28F11 or 34A3, and/or    -   c) binds human TLR3 under acidic conditions, and in particular        under conditions representative of that encountered in an        acidified subcellular compartment of a cell of a cell, e.g.        about pH 5.6, between about pH 4.5 and about 6.5; optionally        wherein the affinity for TLR3 under acidic conditions is not        substantially different, e.g. reduced, compared to binding under        neutral conditions.

Optionally, the method comprises testing an antibody according tosubsteps (a) and (b), substeps (a) and (c), substeps (b) and (c) orsubsteps (a), (b) and (c).

Optionally the method further comprises a step of selecting the antibodyif it is determined to inhibit TLR3 signaling, if it is determined tocompete for binding to a TLR3 polypeptide with antibody 31C3, 29H3,23C8, 28F11 or 34A3, and/or if it is determined to bind human TLR3 underacidic conditions and/or without a reduction in affinity compared toneutral conditions. Optionally the antibody is further tested for itsability to modulate, e.g. inhibit, TLR3 signaling in a dendritic cell,and selected if the antibody is determined to modulate TLR3 signaling ina DC. Optionally, the antibody so selected is selected for use in thetreatment or prevention of a disease (e.g. an antibody that inhibitsTLR3 signaling will be used in inflammatory and autoimmune disorders).Optionally, a quantity of the antibody so selected is produced (e.g. ina recombinant host cell).

In another aspect, the present invention provides a method of producingan antibody that specifically binds to TLR3 in a mammalian subject,particularly in a human subject, said method comprising the steps ofgenerating a plurality of antibodies (e.g. by immunizing a non-humanmammal with an immunogen comprising a TLR3 polypeptide); and selectingan antibody from said plurality that:

-   -   a) inhibits TLR3 signaling, optionally without blocking the        binding of a TLR3 ligand (e.g. dsRNA) to a TLR3 polypeptide,        and/or    -   b) competes for binding to a TLR3 polypeptide with antibody        31C3, 29H3, 23C8, 28F11 or 34A3, and/or    -   c) binds human TLR3 under acidic conditions, and in particular        under conditions representative of that encountered in an        acidified subcellular compartment of a cell of a cell, e.g.        about pH 5.6, between about pH 4.5 and about 6.5; optionally        wherein the affinity for TLR3 under acidic conditions is not        substantially different, e.g. reduced, compared to binding under        neutral conditions.

Optionally, the method comprises selecting an antibody according tosubsteps (a) and (b), substeps (a) and (c), substeps (b) and (c) orsubsteps (a), (b) and (c). Optionally the method further comprisesselecting an antibody that has ability to modulate, e.g. inhibit, TLR3signaling in a dendritic cell, and selected if the antibody isdetermined to modulate TLR3 signaling in a DC.

In another aspect, the present invention provides a method of testing anantibody comprising: a) providing a test antibody; and b) assessingwhether said test antibody induces a decrease in the expression of TLR3at the surface of cells, c) if said antibody does not induces a decreasein the expression of TLR3 at the surface of cells, selecting said testantibody as a candidate for the treatment of a disease (e.g. any of thediseases disclosed herein).

In another aspect, the present invention provides a method of testing anantibody comprising: a) providing a test antibody; b) assessing thebinding affinity of said test antibody under acidic conditions, e.g., ata pH of 5.6; and c) if said antibody has a binding affinity in acidicconditions (e.g., subnanomoral affinity, no substantial decrease inaffinity compared to at neutral conditions, etc.), selecting said testantibody as a candidate for the treatment of a disease.

In another aspect, the present invention provides a method of testing anantibody comprising: a) providing a test antibody; b) assessing whethersaid test antibody is able to bind TLR3 protein in the presence of aTLR3 ligand, e.g. dsRNA; and c) if said antibody is able to bind TLR3protein in the presence of a TLR3 ligand, selecting said test antibodyas a candidate for the treatment of a disease.

In another aspect, the present invention provides a method of selectingan antibody comprising: a) providing a test antibody; b) assessingwhether said test antibody is able to bind TLR3 protein in the presenceof inflammatory cytokines, i.e. IFNα; and c) selecting said testantibody as a candidate for the treatment of a disease.

In another aspect, the present invention provides a method of testing anantibody comprising: a) providing a test antibody; b) assessing whethersaid test antibody is capable of being internalized, e.g. by aTLR3-expressing cell; and c) if said antibody is capable of beinginternalized, preferably wherein the antibody rapidly internalized,e.g., within 2 hours, selecting said test antibody as a candidate forthe treatment of a diseases. In one embodiment, the disease is aninflammatory disorder. In one embodiment, the disease is a cancer.

In one embodiment, the antibodies prepared are monoclonal antibodies. Inanother embodiment, the method further comprise a step in which theability of said antibodies to specifically bind to human TLR3polypeptides is assessed. In one embodiment, the ability of theantibodies to bind to other TLR family members is assessed. In anotherembodiment, the method further comprises the step of making fragments orderivatives of the selected monoclonal antibodies. In one embodiment,the fragments or derivatives are selected from the group consisting ofFab, Fab′, Fab′-SH, F (ab′) 2, Fv, diabodies, single-chain antibodyfragment, multispecific antibodies comprising multiple differentantibody fragments, humanized antibodies, and chimeric antibodies. Inanother embodiment, the non-human mammal is a mouse.

In another aspect, the present invention provides a method of treatingor preventing disease in a patient comprising administering a TLR3antibody of the invention to the patient. In another embodiment, themethod further comprises the step of administering to the patient anappropriate additional therapeutic agent, for example particularly whenthe TLR3 antibody inhibits TLR3 signaling, an additional agent can beselected from the group consisting of immunomodulatory agents,corticosteroids, immunosuppressants, antibiotics, anti-inflammatoryagents, and the like. Particularly when the TLR3 antibody isinternalized by a TLR3-expressing cell and/or linked to a toxin orcytotoxic drug in order to eliminate a TLR3-expressing cell (e.g., acancer cell), an additional agent can be selected from the groupconsisting of an anti-cancer agent, a cytotoxic agent, and the like. Inone aspect the invention provides a method for treating or preventing adisease selected from autoimmunity, inflammation, allergy, asthma,infections, osteoporosis, cirrhosis and sepsis, cancer or other diseasescontemplated herein, comprising administering a therapeuticallyeffective amount of an inhibitory TLR3 antibody to a patient in needthereof. The antibody is administered for a time sufficient to treat orprevent said disease.

In one embodiment, the antibodies of the invention can be used indiagnostics assays or more generally any assays to detect TLR3polypeptides in vitro. In one aspect the present invention provides anin vitro method of detecting a TLR3 polypeptide (e.g. in a biologicalsample) comprising bringing a TLR3 polypeptide (e.g. a cell expressing aTLR3 polypeptide, a purified TLR3 polypeptide, a biological sample,etc.) into contact with a monoclonal antibody of the invention, anddetecting binding of the antibody to the TLR3 polypeptide.

One embodiment is an isolated antibody which competes for binding to aTLR3 polypeptide with any one or any combination of monoclonalantibodies 31C3, 29H3, 23C8, 28F11 or 34A3. In some aspects of thisembodiment, the antibody competes for binding to a TLR3 polypeptide withany one or any combination of monoclonal antibodies 31C3, 29H3, 23C8,28F11 or 34A3 under acid conditions. In other aspects of thisembodiment, the antibody competes for binding to a TLR3 polypeptide withany one or any combination of monoclonal antibodies 31C3, 29H3, 23C8,28F11 or 34A3 under neutral conditions. In further aspects, the antibodyis a monoclonal antibody.

One embodiment is a method for treating or preventing a disease whereinthe inhibition of TLR3 signaling pathway is desirable comprisingadministering a therapeutically effective amount of any of theantibodies described herein to a patient in need thereof. In someaspects, the disease is selected from the group consisting ofautoimmunity, inflammation, allergy, asthma, infection, cirrhosis,sepsis, diabetes, and viral infections.

One embodiment is an isolated antibody which is specific for an epitopein the TLR-3 polypeptide that does not undergo substantial change inelectrostatic potential or undergoes less change than a region ofpositive electrostatic potential when neutral conditions are acidifiedsuch that the binding affinity of said antibody remains substantiallyunchanged. In some aspects, the antibody may be in a compositioncomprising a pharmaceutically acceptable carrier. The antibody may beused in a method for treating or preventing a disease wherein theinhibition of TLR3 signaling pathway is desirable comprisingadministering a therapeutically effective amount of the antibody to apatient in need thereof. In some aspects, the disease is selected fromthe group consisting of autoimmunity, inflammation, allergy, asthma,infection, cirrhosis, sepsis, diabetes, and viral infections.

One embodiment is an isolated antibody which specifically binds to thesame TLR3 epitope as any one or any combination of monoclonal antibodies31C3, 29H3, 23C8, 28F11 or 34A3. In some aspects, the antibody may be ina composition comprising a pharmaceutically acceptable carrier. Theantibody may be used in a for treating or preventing a disease whereinthe inhibition of TLR3 signaling pathway is desirable comprisingadministering a therapeutically effective amount of the antibody to apatient in need thereof. In some aspects, the disease is selected fromthe group consisting of autoimmunity, inflammation, allergy, asthma,infection, cirrhosis, sepsis, diabetes, and viral infections.

These and additional advantageous aspects and features of the inventionmay be further described elsewhere herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C show the inhibition of TLR3 activation markers on myeloidDC. FIG. 1A: level of CD86 expression, FIG. 1B: IL-6 secretion, FIG. 1C:IP-10 secretion. All figures show that compared to control, the antibody31C3 inhibits TLR3 ligand-induced response.

FIGS. 2A and 2B show the inhibition of TLR3 activation markers on MdDC.FIG. 2A: level of CD86 expression, FIG. 2B: IP-10 secretion. All figuresshow that compared to control, the antibody 29H3 inhibits the TLR3ligand-induced response.

FIGS. 3A and 3B show the inhibition of TLR3 activation markers onmyeloid DC, comparing F(ab)′2 fragments of antibody 31C3 to purifiedwhole 31C3 antibodies (indicated by “Pur”). FIG. 3A: level of CD86expression, FIG. 3B: IP-10 secretion. The figures show that F(ab)′2fragments of antibody 31C3 inhibit TLR3 ligand-induced response as wellas purified whole 31C3 antibodies.

FIGS. 4A and 4B show the inhibition of TLR3 activation markers on MdDC,comparing F(ab)′2 fragments of antibody 29H3 to purified whole 29H3antibodies (indicated by “Pur”). FIG. 4A: level of CD86 expression, FIG.4B: IP-10 secretion. The figures show that F(ab)′2 fragments of antibody29H3 inhibit TLR3 ligand-induced response as well as purified whole 29H3antibodies.

FIG. 5 shows a comparison of binding affinity that the antibodiesaccording to the present invention have a stronger binding for the TLR3chip than commercially available antibodies.

FIG. 6 shows the binding of the antibodies according to the invention ona TLR3 chip in the presence or in the absence of polyAU, a ligand forTLR3 receptors. The figure shows that the binding of the antibodies ofthe invention do not block dsRNA binding to the TLR3 dsRNA fixationsite.

FIG. 7 shows that the presence of 29H3 on the TLR3 chip blocks thebinding of 31C3 and vice versa. These results tend to show that bothantibodies compete for an overlapping or highly similar epitope.

FIG. 8 shows molecular surface maps of the extracellular domains of thehuman TLR3 protein, generated by computer modeling.

FIG. 9 shows results of an assay for inhibition of TLR3 signaling in aluciferase based reporter gene activity (293T-TLR3-ISRE), where foldincrease in luciferase is indicated as a function of polyAU dose.Briefly, dsRNA TLR3 agonists were used to induce TLR3 signaling in thereporter assay in the presence of anti-TLR3 antibody 31C3, and TLR3signaling was assessed. Antibody 31C3 strongly inhibited TLR3 signalingin a dose dependent fashion, compared to a control anti-TLR3 antibodyhaving no TLR3 inhibiting activity.

FIG. 10A shows the dose dependant inhibition of TLR3 signalling using a293T-TLR3 luciferase assay, with the commercial TLR3.7 antibody (blackdots), 28F11 (open triangles), 23C8 (open squares) and 31C3 (blacksquares) antibodies according to the invention, compared to a control(no Ab: open dots). FIG. 10B shows the same results in an assaycomparing 31C3 (black squares), 23C8 and 34A3 (black triangles)antibodies.

FIGS. 11A-11C show the effect of the 31C3 (open squares) or 23C8 (blackfull squares) antiTLR3 antibodies in different conditions compared to noantibody (black dots). IP-10 secretion is indicated in ng/ml inordinates, the dose of dsRNA added is indicated in μg/ml in axis. FIG.11A represents the inhibition of TLR3 signaling, in the standardconditions (no preactivation). FIG. 11B represents inhibition of TLR3signaling, with a polyAU prestimulation. FIG. 11C represents inhibitionof TLR3 signaling with an IFNα prestimulation.

FIGS. 12A and 12B show the results the kinetics assays. FIG. 12Arepresents the IP-10 secretion in ng/ml (depending on the polyAU doses)for the 31C3 antibody. FIG. 12B represents the results for the 23C8antibody. The TLR3 mAb is added in the medium either 1 h30 before (blackcrosses), together with (black plus “+”), or 1 h30 after (open squares)the dsRNA, the dsRNA alone (black dots) is provided as a positivecontrol).

FIG. 13 shows specific recognition of human TLR3 by 31C3 mAb in TLR3expressing cells, in vitro. Histogram profile for intracellular FACSstaining with 31C1 mAb are shown for HEK 293T control cells and 293Tcells transfected with human TLR3. Unstained represents the FluorescenceIntensity range obtained with control isotype IgG1

FIGS. 14A and 14B show the inhibition of TLR3-induced activation markersand cytokine secretion on myeloid DC. FIG. 14A: IP-10 secretion, FIG.14B: level of CD86 expression. All figures show that compared tocontrol, the antibody 31C3 (black dots), 28F11 (black triangles) and23C8 (black squares) (here shown at a dose of 50 μg/ml) inhibit TLR3ligand-induced response.

FIGS. 15A and 15B show the binding affinity of the antibodies accordingto the invention. FIG. 15A shows that the antibodies according to thepresent invention have a stronger binding for the TLR3 chip thancommercially available (i.e. TLR3.7) antibodies. FIG. 15B shows thebinding of the antibodies according to the invention on a TLR3 chip whensaid chip has been previously saturated with the 31C3 antibody. Acomparison of binding levels as set forth in FIGS. 15A and 15B underlinethat the antibodies according to the invention have an impaired bindingto hTLR3 when the chip has previously been saturated with the 31C3antibody, on the contrary, the commercial TLR3.7 antibody retains thesame binding level in the presence or in the absence of 31C3 antibody.

FIGS. 16A and 16B show the binding of the 28F11, 34A3 and 23C8antibodies according to the invention on a TLR3 chip in the presence orin the absence of polyAU, a ligand for TLR3 receptors. The figures showthat the binding of the antibodies of the invention do not block dsRNAbinding to the TLR3 dsRNA fixation site.

FIG. 17 shows the binding of the 34A3 antibody, either alone on rhTLR3(bold line or on a chip saturated with 31C3 (dotted line). The figureshows that the two antibodies compete with 31C3 for binding to hTLR3.

FIGS. 18A and 18B show the phylogenetic trees of the CDRs of theantibodies according to the invention. FIG. 18A shows the phylogenetictree for the light chains CDRs and FIG. 18B shows the phylogenetic treefor the heavy chains CDRs. The figures show that there is a high CDRhomology between antibodies 28F11 (28.2), 31C3 (31) and 23C8(23), andthat 23H3 (29) and 34A3 (34) have more differences in amino acidsequences.

FIGS. 19A and 19B show the inhibition of TLR3 activation markers onmyeloid DC by the 34A3 antibody. FIG. 19A: IP-10 secretion, FIG. 19B:IL-6 secretion. All figures show that compared to control (no Ab—opendots) and 31C3 (black squares, as positive control), the antibody 34A3(black triangles) inhibits TLR3 ligand-induced response.

FIGS. 20A and 20B show the FACS analysis of internalization assays asdescribed in example 8. FIG. 20A represents the negative control,representing standard fluorescence of the 293T-ISRE/TLR3 cells in theabsence of an antibody linking TLR3 proteins. FIG. 20B is the positivecontrol, indicating the level of TLR3 expression in 293T-ISRE/TLR3 celllines. FIGS. 20C and 20D represent the proportion of TLR3 proteinscoupled with the 31C3 antibody after 24 h or 2 h incubation,respectively. FIGS. 20D and 20E, showing a similar fluorescence thanFIG. 20B confirm that the binding of TLR3 by antibody 31C3 does notdown-modulate the expression of TLR3 on 293T-ISRE/TLR3 cell lines.

DETAILED DESCRIPTION OF THE INVENTION Introduction

The present invention provides novel methods for producing and usingantibodies and particularly TLR3-modulating antibodies suitable for theprophylaxis and treatment of disorders such as autoimmunity,inflammation, allergy, asthma, cirrhosis, osteoporosis, infection,cancers and sepsis. Antibodies, antibody derivatives, antibodyfragments, and cells producing them are encompassed, as are methods ofproducing the same and methods of treating or diagnosing patients usingthe antibodies and compounds.

While high affinity is desirable in antibodies, generally all antibodiesreported to have affinities in the high picomolar to low nanomolar rangehave been affinity matured in vitro. The scientific literature hasproposed that there is an in vivo affinity ceiling at 100 pM and thatthis might arise because B cells producing antibodies with affinitiesfor antigen above the estimated ceiling would have no selectiveadvantage during normal immune responses. However, examples of highaffinity antibodies exist, including the anti-TNF-alpha antibody“TSK114” which binds to human TNF-alpha with a binding affinity (K(D))of approximately 5.3 pM, which was stated to be about 1,000- and100-fold higher than those of clinically relevant infliximab (Remicade)and adalimumab (Humira) mAbs (Song et al. (2008) Exp. Mol. Med. 40(1):35-42. It is possible that obtaining high affinity antibodies depends onthe antigen; yet the TLR3 antibodies available to date have at bestshown nanomolar affinity. The present anti-TLR3 antibodies howeverdemonstrated very high affinity (as high as 10 picomolar and better than100 picomolar, including for two antibodies that maintained suchaffinity at both neutral and acid conditions).

The present invention is based, at least in part, on the discovery ofmonoclonal antibodies that specifically and efficiently bind TLR3 underacidic conditions corresponding to that encountered in an acidifiedendosomic compartment. Among numerous antibodies assessed, certainantibodies emerged that retained binding to TLR3 at high affinitiesunder acidic conditions, while other antibodies such as those availablecommercially and others selected for TLR3 binding or TLR3 modulationlost affinity despite initially displaying higher (e.g. 2-log₁₀ higher)affinity for TLR3, and/or had low affinity even under neutralconditions. Acidic conditions used were pH 5.6 which is similar to thatobserved in an acidified endosomic compartment, corresponding to theconditions under which TLR3 signaling in inflammatory conditions isbelieved to take place.

Acidic conditions are generally known to affect the structure ofproteins as well as to affect protein-protein interactions. It is known,for example, that MHC class II peptides that are not bound to otherpeptides are rapidly degraded in the acidic conditions of the endosome.However, in the present case the antibodies that lost their high bindingaffinity to immobilized TLR3 under acidic conditions of pH 5.6 had beenpreviously purified under acidic conditions (pH 3). Without wishing tobe bound by theory, this suggests that the loss of binding affinityarose not from inherent instability (degradation) of the antibody atacid conditions, but rather from modifications in the interactionbetween the antibodies and their target antigens.

Modifications in antibody-TLR3 interactions arising from changes in pHare believed to affect interactions of dsRNA with TLR3, since the TLR3ligand poly(I-C) binds and activates TLR3 only at acidic pH. Studieshave reported that poly(I-C) (and other dsRNA) bind TLR3 in a region ofTLR3 of positive electrostatic potential at neutral pH that can undergoa change in electrostatic potential in acidic conditions (that is,acidic conditions in the range of pH 4.5 to 6.5, or around 5.6). Thepresent antibodies, however, are believed to bind an epitope that doesnot undergo substantial change in electrostatic potential (or undergoesless change than e.g. a region of positive electrostatic potential) whenconditions are acidified such that the binding affinity of theantibodies remains substantially unchanged. This can, in one aspect,manifest itself in terms of affinity of the antibodies for TLR3, sincethe antibodies do not have substantially different (lower and/or higher)affinity for binding human TLR3 under acidic conditions than underneutral conditions e.g. where the K_(D) for binding to TLR3 differs byno more than 0.2-, 0.3-, 0.4-, 0.5-, 1.0-, or 1.5-log₁₀. The K_(D) forbinding to TLR3 under acid and neutral conditions differed by less than0.5-log₁₀ for antibodies 31C3 and 29H7. It is believed that the epitopeto which the antibodies of the invention bind may have negativeelectrostatic potential at neutral pH. Regions of negative, positive orneutral electrostatic potential on the surface of the TLR3 protein areshown in FIG. 8 or in also shown in FIG. 5D of Choe et al. (2005)Science 309:581-585, the disclosure of which is incorporated herein byreference. Whereas antibodies that inhibit TLR3 by interfering withbinding of dsRNA ligands to TLR3 will be likely to bind to a region ofpositive or neutral electrostatic potential near the C-terminal on theglycosylation-free face of TLR3 and therefore bind in a region of TLR3that can undergo a greater change in electrostatic potential, thepresent antibodies appear to bind to a region in TLR3 not involved inbinding to dsRNA ligands, while nevertheless retaining the ability toinhibit signaling by the TLR3 protein, e.g. by inhibiting TLR3 fromadopting a conformation required for ultimately transducing a signal.

The present invention is also based, at least in part, on the discoveryof high affinity monoclonal antibodies that specifically and efficientlyinhibit the TLR3 signaling pathway. The inventors have identifiedepitopes present on human TLR3, including the epitope recognized byantibody 31C3, 29H3, 23C8, 28F11 or 34A3, which are particularlyefficient in inhibiting TLR3 signaling, and inhibiting cytokine releasein response to stimulation with a TLR3 ligand.

The antibodies of the invention that inhibit TLR3 signaling will beparticularly useful in treating and/or preventing autoimmune diseases,inflammatory disease and other diseases where inhibiting TLR3 signalingis beneficial. Autoimmune and inflammatory diseases arise from anoveractive immune response of the body against substances and tissuesnormally present in the body. In both autoimmune and inflammatorydiseases the condition arises through aberrant reactions of the humanadaptive or innate immune systems. In autoimmunity, the patient's immunesystem is activated against the body's own proteins. In inflammatorydiseases (including in infection which can lead to inflammatoryconditions), it is the overreaction of the immune system, and itssubsequent downstream signaling (TNF, IFN, etc), which causes problems.Autoimmune diseases result from the propagation of T and B cells thatrecognize self antigens and mediate tissue destruction. Viral infectionshave long been suspected to instigate or overtly precipitateautoimmunity. In Lang et al. (J. Clin. Invest. 116:2456-2463, 2006) ithas been demonstrated that viruses can initiate autoimmune damagethrough yet another mechanism.

It has been recently established that dsRNA are ligands for TLR3(Alexoupoulou et al. (2001), Nature 413: 732-738), it has also been morerecently shown that RNA released from either damaged tissue or tissuescould also act as TLR3 ligands (Kariko et al, (2004) J. Biol. Chem.).Inappropriate activation of the TLRs by their endogenous RNA ligandswithin immune complexes almost certainly is an important factorcontributing to pathogenesis of various inflammatory and autoimmunediseases. Recent papers suggest a role of TLR3 in inflammatory diseases(Cavassani et al. 2008), as TLR3 ligands amplify the hyperinflammatoryresponse observed during sepsis, or autoimmunity diseases, such asrheumatoid arthritis (Bokarewa et al. (2008) Eur J. Immunol.), systemiclupus erythematosus (Rahman et al., (2006) Springer Sem. inImmunopathol.), and diabetes (Nature Med. 2005). It has also beenreported that TLR3 may play a detrimental role in viral infections suchas Western Reserve vaccinia virus, where TLR3 contributes to viralreplication, detrimental lung inflammation and recruitment of leukocytesto the lung, resulting in increased morbidity or in West Nile Virus(WNV) where TLR3 allows the virus to cross the blood-brain barrier (BBB)and cause lethal encephalitis. The antibodies of the present inventionthat inhibit TLR3 under pH conditions corresponding to that of endosomes(e.g. as in myeloid DC) will be useful in the treatment and preventionof these conditions, including but not limited to the viral infectionsthemselves and the conditions (e.g. autoimmune or inflammatoryconditions) caused or enhanced by viral infection.

Zorde-Khvalevsky et al. (2009) Hepatology 49 report that hepatocyteproliferation was accelerated following partial hepatectomy in theabsence of TLR3, while the levels of IL-6 and soluble interleukin-6receptor (sIL-6R) were significantly lower, and further that afterpartial hepatectomy TLR3 signaling is induced in hepatocytes, resultingin activation of NF-kB, and that the presence of active TLR3 in Kupffercells inhibits NF-kB activation. TLR3 signaling was therefore found toattenuate the initiation of liver regeneration; the anti-TLR3 antibodiesof the invention can therefore be used in a method of inducing liverregeneration, in particular to treat and prevent diseases involvingliver damage, e.g. cirrhosis, or diseases that are known to give rise tosuch liver damage such as alcoholism, hepatitis B or C infection orfatty liver diseases.

Kim et al. (2009) Immunol. Lett. report that TLR3 promotesosteoclastogenesis in the RA synovium both directly and indirectly bystimulating human monocytes directly to promote osteoclastdifferentiation and by inducing RANKL expression indirectly in RA-FLS.The expression of RANKL promotes the differentiation of osteoclasts inthe RA synovium, and anti-RANKL antibodies (denosumab, Amgen Inc.) areefficacious in the treatment of osteoporosis. The anti-TLR3 antibodiesof the invention can therefore be use to treat and prevent inflammatorybone destruction, e.g. osteoporosis, particularly in RA patients.

Wen et al. (2004) J. Immunol. 172: 3172-3180 suggest that autoimmunediseases can be induced by a viral-like stimulus, and identify TLR3 ascapable of mediating such induction. Results demonstrate that polyICtogether with insulin, but not insulin alone or other TLR ligands (CpG,LPS, PGN), can induce autoimmune diabetes and apoptosis of pancreaticislets in a mouse model. Furthermore, TLR3 showed the highest expressionlevel in all individuals, compared to other TLRs. The anti-TLR3antibodies of the invention can therefore be use to treat and preventdiabetes and islet autoimmunity.

The antibodies of the present invention that bind TLR3 under acidicconditions will generally bind both cell surface TLR3 and endosomic TLR3at high affinity, such that the antibodies will be useful in anysituation (e.g. treatment or prevention of disease) where targeting(e.g. modulating) TLR3 is useful. TLR3 has been found in some cases ofinflammation the surface of macrophages and blocking TLR3 uponchloroquine neutralization of endosomal acification neverthelessexhibited some anti-inflammatory activity (Cavassani et al. 2008,supra). However, the antibodies of the invention will have the greatestadvantage over other antibodies in the treatment or prevention ofdiseases where the modulating (e.g. inhibiting) the signaling by TLR3 inthe cytosolic (e.g. endosomic) compartments is useful or required, andthe relative importance of modulating signaling of such compartmentsTLR3 may depend on the disease. One example of such as disease isrheumatoid arthritis; endosomic compartment-expressed TLR3 is believedto play an important role in rheumatoid arthritis, since treatment withchloroquine, an inhibitor of endosomal acidification, inhibits TLR3signaling and inhibits production of inflammatory cytokines fromsynovial cultures from patients having rheumatoid arthritis (Sacre etal. (2008) J. Immunol. 181:8002-8009). Endosomic compartment-expressedTLR3 is believed to play an important role in a number of other diseaseswhere DC (e.g. myeloid DC) are involved in exacerbating disease, as mDChave a well documented capacity to take up antigens from apoptotic ornecrotic cells including during tissue necrosis during acuteinflammation.

Since the present antibodies are specific for TLR3, they can also beused for other purposes, including purifying TLR3 or TLR3-expressingcells, modulating (e.g. activating or inhibiting) TLR3 receptors invitro, ex vivo, or in vivo, targeting TLR3-expressing cells fordestruction in vivo, or specifically labeling/binding TLR3 in vivo, exvivo, or in vitro, including for methods such as immunoblotting, IHCanalysis, i.e. on frozen biopsies, FACS analysis, andimmunoprecipitation.

DEFINITIONS

As used herein, “TLR3 ligands” refer to any compound that canspecifically bind to and alter the activity of TLR3 in vitro, ex vivo,or in vivo. The compound can be a naturally occurring ligand, e.g.,generally dsRNA or viral dsRNA, or a synthetic ligand such as polyIC orpolyAU. The compound can be any type of molecule, including inorganic ororganic compounds or elements, including proteins (such as antibodies),nucleic acids, carbohydrates, lipids, or any other molecular entity.Further, such compounds can modulate TLR3 receptors in any way,including activating or inhibiting, and by any mechanism, including bybinding to the receptor and triggering or shutting off activity in amanner similar to a naturally occurring ligand, or by binding to thereceptor and blocking access to other ligands. Preferably, the ligandactivates the receptor, and as such can be used to induce the productionof cytokines by TLR3-expressing cells.

The term “antibody,” as used herein, refers to polyclonal and monoclonalantibodies. Depending on the type of constant domain in the heavychains, antibodies are assigned to one of five major classes: IgA, IgD,IgE, IgG, and IgM. Several of these are further divided into subclassesor isotypes, such as IgG1, IgG2, IgG3, IgG4, and the like. An exemplaryimmunoglobulin (antibody) structural unit comprises a tetramer. Eachtetramer is composed of two identical pairs of polypeptide chains, eachpair having one “light” (about 25 kDa) and one “heavy” chain (about50-70 kDa). The N-terminus of each chain defines a variable region ofabout 100 to 110 or more amino acids that is primarily responsible forantigen recognition. The terms variable light chain (V_(L)) and variableheavy chain (V_(H)) refer to these light and heavy chains respectively.The heavy-chain constant domains that correspond to the differentclasses of immunoglobulins are termed “alpha,” “delta,” “epsilon,”“gamma” and “mu,” respectively. The subunit structures andthree-dimensional configurations of different classes of immunoglobulinsare well known. IgG and/or IgM are the preferred classes of antibodiesemployed in this invention, with IgG being particularly preferred,because they are the most common antibodies in the physiologicalsituation and because they are most easily made in a laboratory setting.Preferably the antibody of this invention is a monoclonal antibody.Particularly preferred are humanized, chimeric, human, orotherwise-human-suitable antibodies. “Antibodies” also includes anyfragment or derivative of any of the herein described antibodies.

The term “specifically binds to” means that an antibody can bindpreferably in a competitive binding assay to the binding partner, e.g.TLR3, as assessed using either recombinant forms of the proteins,epitopes therein, or native proteins present on the surface of isolatedtarget cells. Competitive binding assays and other methods fordetermining specific binding are further described below and are wellknown in the art.

When an antibody is said to “compete with” a particular monoclonalantibody (e. g. 31C3, 29H3, 23C8, 28F11 or 34A3), it means that theantibody competes with the monoclonal antibody in a binding assay usingeither recombinant TLR3 molecules or surface expressed TLR3 molecules.For example, if a test antibody reduces the binding of 31C3, 29H3, 23C8,28F11 or 34A3 to a TLR3 polypeptide or TLR3-expressing cell in a bindingassay, the antibody is said to “compete” respectively with 31C3, 29H3,23C8, 28F11 or 34A3.

The term “affinity”, as used herein, means the strength of the bindingof an antibody to an epitope. The affinity of an antibody is given bythe dissociation constant Kd, defined as [Ab]×[Ag]/[Ab-Ag], where[Ab-Ag] is the molar concentration of the antibody-antigen complex, [Ab]is the molar concentration of the unbound antibody and [Ag] is the molarconcentration of the unbound antigen. The affinity constant K_(a) isdefined by 1/Kd. Preferred methods for determining the affinity of mAbscan be found in Harlow, et al., Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1988), Coliganet al., eds., Current Protocols in Immunology, Greene Publishing Assoc.and Wiley Interscience, N.Y., (1992, 1993), and Muller, Meth. Enzymol.92:589-601 (1983), which references are entirely incorporated herein byreference. One preferred and standard method well known in the art fordetermining the affinity of mAbs is the use of Biacore instruments.

Within the context of this invention a “determinant” designates a siteof interaction or binding on a polypeptide.

The term “epitope” is defined as an antigenic determinant, and is thearea or region on an antigen to which an antibody binds. A proteinepitope may comprise amino acid residues directly involved in thebinding as well as amino acid residues which are effectively blocked bythe specific antigen binding antibody or peptide, i.e., amino acidresidues within the “footprint” of the antibody. It is the simplest formor smallest structural area on a complex antigen molecule that cancombine with e.g., an antibody or a receptor. Epitopes can be linear orconformational/structural. The term “linear epitope” is defined as anepitope composed of amino acid residues that are contiguous on thelinear sequence of amino acids (primary structure). The term“conformational or structural epitope” is defined as an epitope composedof amino acid residues that are not all contiguous and thus representseparated parts of the linear sequence of amino acids that are broughtinto proximity to one another by folding of the molecule (secondary,tertiary and/or quaternary structures). A conformational epitope isdependent on the 3-dimensional structure. The term ‘conformational’ istherefore often used interchangeably with ‘structural’.

By “immunogenic fragment,” it is herein meant any polypeptidic orpeptidic fragment that is capable of eliciting an immune response suchas (i) the generation of antibodies binding said fragment and/or bindingany form of the molecule comprising said fragment, including themembrane-bound receptor and mutants derived therefrom, (ii) thestimulation of a T-cell response involving T-cells reacting to thebi-molecular complex comprising any MHC molecule and a peptide derivedfrom said fragment, (iii) the binding of transfected vehicles such asbacteriophages or bacteria expressing genes encoding mammalianimmunoglobulins. Alternatively, an immunogenic fragment also refers toany construction capable of eliciting an immune response as definedabove, such as a peptidic fragment conjugated to a carrier protein bycovalent coupling, a chimeric recombinant polypeptide constructcomprising said peptidic fragment in its amino acid sequence, andspecifically includes cells transfected with a cDNA of which sequencecomprises a portion encoding said fragment.

“Toxic” or “cytotoxic” peptides or small molecules encompass anycompound that can slow down, halt, or reverse the proliferation ofcells, decrease their activity in any detectable way, or directly orindirectly kill them. Preferably, toxic or cytotoxic compounds work bydirectly killing the cells, by provoking apoptosis or otherwise. As usedherein, a toxic “peptide” can include any peptide, polypeptide, orderivative of such, including peptide- or polypeptide-derivatives withunnatural amino acids or modified linkages. A toxic “small molecule” canincludes any toxic compound or element, preferably with a size of lessthan 10 kD, 5 kD, 1 kD, 750 D, 600 D, 500 D, 400 D, 300 D, or smaller.

A “human-suitable” antibody refers to any antibody, derivatizedantibody, or antibody fragment that can be safely used in humans for,e.g. the therapeutic methods described herein. Human-suitable antibodiesinclude all types of humanized, chimeric, or fully human antibodies, orany antibodies in which at least a portion of the antibodies is derivedfrom humans or otherwise modified so as to avoid the immune responsethat is generally provoked when native non-human antibodies are used.

For the purposes of the present invention, a “humanized” or “human”antibody refers to an antibody in which the constant and variableframework region of one or more human immunoglobulins is fused with thebinding region, e.g. the CDR, of an animal immunoglobulin. Suchantibodies are designed to maintain the binding specificity of thenon-human antibody from which the binding regions are derived, but toavoid an immune reaction against the non-human antibody. Such antibodiescan be obtained from transgenic mice or other animals that have been“engineered” to produce specific human antibodies in response toantigenic challenge (see, e.g., Green et al. (1994) Nature Genet 7:13;Lonberg et al. (1994) Nature 368:856; Taylor et al. (1994) Int Immun6:579, the entire teachings of which are herein incorporated byreference). A fully human antibody also can be constructed by genetic orchromosomal transfection methods, as well as phage display technology,all of which are known in the art (see, e.g., McCafferty et al. (1990)Nature 348:552-553). Human antibodies may also be generated by in vitroactivated B cells (see, e.g., U.S. Pat. Nos. 5,567,610 and 5,229,275,which are incorporated in their entirety by reference).

A “chimeric antibody” is an antibody molecule in which (a) the constantregion, or a portion thereof, is altered, replaced or exchanged so thatthe antigen binding site (variable region) is linked to a constantregion of a different or altered class, effector function and/orspecies, or an entirely different molecule which confers new propertiesto the chimeric antibody, e.g., an enzyme, toxin, hormone, growthfactor, drug, etc.; or (b) the variable region, or a portion thereof, isaltered, replaced or exchanged with a variable region having a differentor altered antigen specificity.

The terms “Fc domain,” “Fc portion,” and “Fc region” refer to aC-terminal fragment of an antibody heavy chain, e.g., from about aminoacid (aa) 230 to about aa 450 of human γ (gamma) heavy chain or itscounterpart sequence in other types of antibody heavy chains (e.g., α,δ, ε and μ for human antibodies), or a naturally occurring allotypethereof. Unless otherwise specified, the commonly accepted Kabat aminoacid numbering for immunoglobulins is used throughout this disclosure(see Kabat et al. (1991) Sequences of Protein of Immunological Interest,5th ed., United States Public Health Service, National Institute ofHealth, Bethesda, Md.).

The terms “isolated”, “purified” or “biologically pure” refer tomaterial that is substantially or essentially free from components whichnormally accompany it as found in its native state. Purity andhomogeneity are typically determined using analytical chemistrytechniques such as polyacrylamide gel electrophoresis or highperformance liquid chromatography. A protein that is the predominantspecies present in a preparation is substantially purified.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymer.

The term “recombinant” when used with reference, e.g., to a cell, ornucleic acid, protein, or vector, indicates that the cell, nucleic acid,protein or vector, has been modified by the introduction of aheterologous nucleic acid or protein or the alteration of a nativenucleic acid or protein, or that the cell is derived from a cell somodified. Thus, for example, recombinant cells express genes that arenot found within the native (nonrecombinant) form of the cell or expressnative genes that are otherwise abnormally expressed, under expressed ornot expressed at all.

Within the context of this invention, the term antibody that “binds” acommon determinant designates an antibody that binds said determinantwith specificity and/or affinity.

Producing Anti-TLR3 Antibodies

The antibodies of this invention specifically bind TLR3. Antibodies ofthe invention furthermore bind TLR3 under acidic conditionscorresponding to that encountered in an acidified endosomic compartment.Antibodies of the invention are furthermore capable of inhibiting theTLR3 signaling pathway. The ability of the inhibitory antibodies tospecifically inhibit TLR3 signaling pathway makes them useful fornumerous applications, in particular for treating or preventing diseaseswherein the inhibition of TLR3 signaling pathway is desirable, i.e.avoid further cytokine and chemokine secretion as well as cellularactivation, as described herein.

In one embodiment, the invention provides an antibody that binds humanTLR3, and competes for binding to human TLR3 with monoclonal antibody31C3, 29H3, 23C8, 28F11 or 34A3. Antibody 31C3 is produced by the celldeposited as 31C3.1 with the Collection Nationale de Culture deMicroorganismes (CNCM), Institut Pasteur, 25 rue de Docteur Roux,F-75724 Paris on 3 Jul. 2009, under the number CNCM I-4186. Antibody29H3 is produced by the cell deposited as 29H3.7 with the CollectionNationale de Culture de Microorganismes (CNCM), Institut Pasteur, 25 ruede Docteur Roux, F-75724 Paris on 3 Jul. 2009, under the number CNCMI-4187.

“TLR3”, “TLR3 polypeptide” and “TLR3 receptor”, used interchangeably,are used herein to refer to Toll-Like Receptor 3, a member of theToll-like receptor (TLRs) family. The amino acid sequence of human TLR3is shown in SEQ ID NO: 1 (NCBI accession number NP_003256, thedisclosure of which is incorporated herein by reference). The human TLR3mRNA sequence is described in NCBI accession number NM_003265. HumanTLR3 sequences are also described in PCT patent publication no. WO98/50547, the disclosure of which is incorporated herein by reference.

In one aspect, the invention provides an antibody that competes withmonoclonal antibody 31C3, 29H3, 23C8, 28F11 or 34A3 and recognizes,binds to, or has immunospecificity for substantially or essentially thesame, or the same, epitope or “epitopic site” on a TLR3 molecule asmonoclonal antibody 31C3, 29H3, 23C8, 28F11 or 34A3. In otherembodiments, the monoclonal antibody consists of, or is a derivative orfragment of, antibody 31C3, 29H3, 23C8, 28F11 or 34A3.

It will be appreciated that, while preferred antibodies bind to the sameepitope as antibody 31C3, 29H3, 23C8, 28F11 or 34A3, the presentantibodies can recognize and be raised against any part of the TLR3polypeptide. For example, any fragment of TLR3, preferably but notexclusively human TLR3, or any combination of TLR3 fragments, can beused as immunogens to raise antibodies, and the antibodies of theinvention can recognize epitopes at any location within the TLR3polypeptide, so long as they can do so on TLR3 expressing cells such asMdDC or MoDC as described herein. In an embodiment, the recognizedepitopes are present on the cell surface, i.e. they are accessible toantibodies present outside of the cell. Most preferably, the epitope isthe epitope specifically recognized by antibody 31C3, 29H3, 23C8, 28F11or 34A3. Further, antibodies recognizing distinct epitopes within TLR3can be used in combination, e.g. to bind to TLR3 polypeptides withmaximum efficacy and breadth among different individuals.

The antibodies of this invention may be produced by a variety oftechniques known in the art. Typically, they are produced byimmunization of a non-human animal, preferably a mouse, with animmunogen comprising a TLR3 polypeptide, preferably a human TLR3polypeptide. The TLR3 polypeptide may comprise the full length sequenceof a human TLR3 polypeptide, or a fragment or derivative thereof,typically an immunogenic fragment, i.e., a portion of the polypeptidecomprising an epitope exposed on the surface of cells expressing a TLR3polypeptide, preferably the epitope recognized by the 31C3, 29H3, 23C8,28F11 or 34A3 antibody. Such fragments typically contain at least about7 consecutive amino acids of the mature polypeptide sequence, even morepreferably at least about 10 consecutive amino acids thereof. Fragmentstypically are essentially derived from the extra-cellular domain of thereceptor. In a preferred embodiment, the immunogen comprises a wild-typehuman TLR3 polypeptide in a lipid membrane, typically at the surface ofa cell. In a specific embodiment, the immunogen comprises intact cells,particularly intact human cells, optionally treated or lysed. In anotherpreferred embodiment, the polypeptide is a recombinant TLR3 polypeptide.

The step of immunizing a non-human mammal with an antigen may be carriedout in any manner well known in the art for stimulating the productionof antibodies in a mouse (see, for example, E. Harlow and D. Lane,Antibodies: A Laboratory Manual., Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y. (1988), the entire disclosure of which isherein incorporated by reference). The immunogen is suspended ordissolved in a buffer, optionally with an adjuvant, such as complete orincomplete Freund's adjuvant. Methods for determining the amount ofimmunogen, types of buffers and amounts of adjuvant are well known tothose of skill in the art and are not limiting in any way on the presentinvention. These parameters may be different for different immunogens,but are easily elucidated.

Similarly, the location and frequency of immunization sufficient tostimulate the production of antibodies is also well known in the art. Ina typical immunization protocol, the non-human animals are injectedintraperitoneally with antigen on day 1 and again about a week later.This is followed by recall injections of the antigen around day 20,optionally with an adjuvant such as incomplete Freund's adjuvant. Therecall injections are performed intravenously and may be repeated forseveral consecutive days. This is followed by a booster injection at day40, either intravenously or intraperitoneally, typically withoutadjuvant. This protocol results in the production of antigen-specificantibody-producing B cells after about 40 days. Other protocols may alsobe used as long as they result in the production of B cells expressingan antibody directed to the antigen used in immunization.

For polyclonal antibody preparation, serum is obtained from an immunizednon-human animal and the antibodies present therein isolated bywell-known techniques. The serum may be affinity purified using any ofthe immunogens set forth above linked to a solid support so as to obtainantibodies that react with TLR3 polypeptides.

In an alternate embodiment, lymphocytes from a non-immunized non-humanmammal are isolated, grown in vitro, and then exposed to the immunogenin cell culture. The lymphocytes are then harvested and the fusion stepdescribed below is carried out.

For preferred monoclonal antibodies, the next step is the isolation ofsplenocytes from the immunized non-human mammal and the subsequentfusion of those splenocytes with an immortalized cell in order to forman antibody-producing hybridoma. The isolation of splenocytes from anon-human mammal is well-known in the art and typically involvesremoving the spleen from an anesthetized non-human mammal, cutting itinto small pieces and squeezing the splenocytes from the splenic capsulethrough a nylon mesh of a cell strainer into an appropriate buffer so asto produce a single cell suspension. The cells are washed, centrifugedand resuspended in a buffer that lyses any red blood cells. The solutionis again centrifuged and remaining lymphocytes in the pellet are finallyresuspended in fresh buffer.

Once isolated and present in single cell suspension, the lymphocytes canbe fused to an immortal cell line. This is typically a mouse myelomacell line, although many other immortal cell lines useful for creatinghybridomas are known in the art. Preferred murine myeloma lines include,but are not limited to, those derived from MOPC-21 and MPC-11 mousetumors available from the Salk Institute Cell Distribution Center, SanDiego, U.S.A, X63 Ag8653 and SP-2 cells available from the American TypeCulture Collection, Rockville, Md. U.S.A The fusion is effected usingpolyethylene glycol or the like. The resulting hybridomas are then grownin selective media that contains one or more substances that inhibit thegrowth or survival of the unfused, parental myeloma cells. For example,if the parental myeloma cells lack the enzyme hypoxanthine guaninephosphoribosyl transferase (HGPRT or HPRT), the culture medium for thehybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

Hybridomas are typically grown on a feeder layer of macrophages. Themacrophages are preferably from littermates of the non-human mammal usedto isolate splenocytes and are typically primed with incomplete Freund'sadjuvant or the like several days before plating the hybridomas. Fusionmethods are described in Goding, “Monoclonal Antibodies: Principles andPractice,” pp. 59-103 (Academic Press, 1986), the disclosure of which isherein incorporated by reference.

The cells are allowed to grow in the selection media for sufficient timefor colony formation and antibody production. This is usually betweenabout 7 and about 14 days.

The hybridoma colonies are then assayed for the production of antibodiesthat specifically bind to TLR3 polypeptide gene products, optionally theepitope specifically recognized by antibody 31C3, 29H3, 23C8, 28F11 or34A3. The assay is typically a colorimetric ELISA-type assay, althoughany assay may be employed that can be adapted to the wells that thehybridomas are grown in. Other assays include radioimmunoassays orfluorescence activated cell sorting. The wells positive for the desiredantibody production are examined to determine if one or more distinctcolonies are present. If more than one colony is present, the cells maybe re-cloned and grown to ensure that only a single cell has given riseto the colony producing the desired antibody. Typically, the antibodieswill also be tested for the ability to bind to TLR3 polypeptides, e.g.,TLR3-expressing cells, in paraffin-embedded tissue sections, asdescribed below.

Hybridomas that are confirmed to produce a monoclonal antibody of thisinvention can be grown up in larger amounts in an appropriate medium,such as DMEM or RPMI-1640. Alternatively, the hybridoma cells can begrown in vivo as ascites tumors in an animal.

After sufficient growth to produce the desired monoclonal antibody, thegrowth media containing monoclonal antibody (or the ascites fluid) isseparated away from the cells and the monoclonal antibody presenttherein is purified. Purification is typically achieved by gelelectrophoresis, dialysis, chromatography using protein A or proteinG-Sepharose, or an anti-mouse Ig linked to a solid support such asagarose or Sepharose beads (all described, for example, in the AntibodyPurification Handbook, Biosciences, publication No. 18-1037-46, EditionAC, the disclosure of which is hereby incorporated by reference). Thebound antibody is typically eluted from protein A/protein G columns byusing low pH buffers (glycine or acetate buffers of pH 3.0 or less) withimmediate neutralization of antibody-containing fractions. Thesefractions are pooled, dialyzed, and concentrated as needed.

Positive wells with a single apparent colony are typically re-cloned andre-assayed to insure only one monoclonal antibody is being detected andproduced.

Antibodies may also be produced by selection of combinatorial librariesof immunoglobulins, as disclosed for instance in (Ward et al. Nature,341 (1989) p. 544, the entire disclosure of which is herein incorporatedby reference).

The identification of one or more antibodies that bind(s) to TLR3,particularly substantially or essentially the same epitope as monoclonalantibody 31C3, 29H3, 23C8, 28F11 or 34A3, can be readily determinedusing any one of a variety of immunological screening assays in whichantibody competition can be assessed. Many such assays are routinelypracticed and are well known in the art (see, e. g., U.S. Pat. No.5,660,827, issued Aug. 26, 1997, which is specifically incorporatedherein by reference). It will be understood that actually determiningthe epitope to which an antibody described herein binds is not in anyway required to identify an antibody that binds to the same orsubstantially the same epitope as the monoclonal antibody describedherein.

For example, where the test antibodies to be examined are obtained fromdifferent source animals, or are even of a different Ig isotype, asimple competition assay may be employed in which the control (31C3,29H3, 23C8, 28F11 or 34A3, for example) and test antibodies are admixed(or pre-adsorbed) and applied to a sample containing TLR3 polypeptides.Protocols based upon western blotting and the use of BIACORE analysisare suitable for use in such competition studies.

In certain embodiments, one pre-mixes the control antibodies (31C3,29H3, 23C8, 28F11 or 34A3, for example) with varying amounts of the testantibodies (e.g., about 1:10 or about 1:100) for a period of time priorto applying to the TLR3 antigen sample. In other embodiments, thecontrol and varying amounts of test antibodies can simply be admixedduring exposure to the TLR3 antigen sample. As long as one candistinguish bound from free antibodies (e. g., by using separation orwashing techniques to eliminate unbound antibodies) and 31C3, 29H3,23C8, 28F11 or 34A3 from the test antibodies (e. g., by usingspecies-specific or isotype-specific secondary antibodies or byspecifically labeling 31C3, 29H3, 23C8, 28F11 or 34A3 with a detectablelabel) one can determine if the test antibodies reduce the binding of31C3, 29H3, 23C8, 28F11 or 34A3 to the antigens, indicating that thetest antibody recognizes substantially the same epitope as 31C3, 29H3,23C8, 28F11 or 34A3. The binding of the (labeled) control antibodies inthe absence of a completely irrelevant antibody can serve as the controlhigh value. The control low value can be obtained by incubating thelabeled (31C3, 29H3, 23C8, 28F11 or 34A3) antibodies with unlabelledantibodies of exactly the same type (31C3, 29H3, 23C8, 28F11 or 34A3),where competition would occur and reduce binding of the labeledantibodies. In a test assay, a significant reduction in labeled antibodyreactivity in the presence of a test antibody is indicative of a testantibody that recognizes substantially the same epitope, i.e., one that“cross-reacts” or competes with the labeled (31C3, 29H3, 23C8, 28F11 or34A3) antibody. Any test antibody that reduces the binding of 31C3,29H3, 23C8, 28F11 or 34A3 to TLR3 antigens by at least about 50%, suchas at least about 60%, or more preferably at least about 80% or 90% (e.g., about 65-100%), at any ratio of 31C3, 29H3, 23C8, 28F11 or 34A3:testantibody between about 1:10 and about 1:100 is considered to be anantibody that binds to substantially the same epitope or determinant as31C3, 29H3, 23C8, 28F11 or 34A3. Preferably, such test antibody willreduce the binding of 31C3, 29H3, 23C8, 28F11 or 34A3 to the TLR3antigen by at least about 90% (e.g., about 95%).

Competition can also be assessed by, for example, a flow cytometry test.In such a test, cells bearing a given TLR3 polypeptide can be incubatedfirst with 31C3, 29H3, 23C8, 28F11 or 34A3, for example, and then withthe test antibody labeled with a fluorochrome or biotin. The antibody issaid to compete with 31C3, 29H3, 23C8, 28F11 or 34A3 if the bindingobtained upon preincubation with a saturating amount of 31C3, 29H3,23C8, 28F11 or 34A3 is about 80%, preferably about 50%, about 40% orless (e.g., about 30%, 20% or 10%) of the binding (as measured by meanof fluorescence) obtained by the antibody without preincubation with31C3, 29H3, 23C8, 28F11 or 34A3. Alternatively, an antibody is said tocompete with 31C3, 29H3, 23C8, 28F11 or 34A3 if the binding obtainedwith a labeled 31C3, 29H3, 23C8, 28F11 or 34A3 antibody (by afluorochrome or biotin) on cells preincubated with a saturating amountof test antibody is about 80%, preferably about 50%, about 40%, or less(e. g., about 30%, 20% or 10%) of the binding obtained withoutpreincubation with the test antibody.

A simple competition assay in which a test antibody is pre-adsorbed andapplied at saturating concentration to a surface onto which a TLR3antigen is immobilized may also be employed. The surface in the simplecompetition assay is preferably a BIACORE chip (or other media suitablefor surface plasmon resonance analysis). The control antibody (e.g.,31C3, 29H3, 23C8, 28F11 or 34A3) is then brought into contact with thesurface at a TLR3-saturating concentration and the TLR3 and surfacebinding of the control antibody is measured. This binding of the controlantibody is compared with the binding of the control antibody to theTLR3-containing surface in the absence of test antibody. In a testassay, a significant reduction in binding of the TLR3-containing surfaceby the control antibody in the presence of a test antibody indicatesthat the test antibody recognizes substantially the same epitope as thecontrol antibody such that the test antibody “cross-reacts” with thecontrol antibody. Any test antibody that reduces the binding of control(such as 31C3, 29H3, 23C8, 28F11 or 34A3) antibody to a TLR3 antigen byat least about 30% or more, preferably about 40%, can be considered tobe an antibody that binds to substantially the same epitope ordeterminant as a control (e.g., 31C3, 29H3, 23C8, 28F11 or 34A3).Preferably, such a test antibody will reduce the binding of the controlantibody (e.g., 31C3, 29H3, 23C8, 28F11 or 34A3) to the TLR3 antigen byat least about 50% (e. g., at least about 60%, at least about 70%, ormore). It will be appreciated that the order of control and testantibodies can be reversed: that is, the control antibody can be firstbound to the surface and the test antibody is brought into contact withthe surface thereafter in a competition assay. Preferably, the antibodyhaving higher affinity for the TLR3 antigen is bound to the surfacefirst, as it will be expected that the decrease in binding seen for thesecond antibody (assuming the antibodies are cross-reacting) will be ofgreater magnitude. Further examples of such assays are provided in,e.g., Saunal (1995) J. Immunol. Methods 183: 33-41, the disclosure ofwhich is incorporated herein by reference.

Determination of whether an antibody binds within an epitope region canbe carried out in ways known to the person skilled in the art. As oneexample of such mapping/characterization methods, an epitope region foran anti-TLR3 antibody may be determined by epitope “foot-printing” usingchemical modification of the exposed amines/carboxyls in the TLR3protein. One specific example of such a foot-printing technique is theuse of HXMS (hydrogen-deuterium exchange detected by mass spectrometry)wherein a hydrogen/deuterium exchange of receptor and ligand proteinamide protons, binding, and back exchange occurs, wherein the backboneamide groups participating in protein binding are protected from backexchange and therefore will remain deuterated. Relevant regions can beidentified at this point by peptic proteolysis, fast microborehigh-performance liquid chromatography separation, and/or electrosprayionization mass spectrometry. See, e. g., Ehring H, AnalyticalBiochemistry, Vol. 267 (2) pp. 252-259 (1999) Engen, J. R. and Smith, D.L. (2001) Anal. Chem. 73, 256A-265A. Another example of a suitableepitope identification technique is nuclear magnetic resonance epitopemapping (NMR), where typically the position of the signals intwo-dimensional NMR spectra of the free antigen and the antigencomplexed with the antigen binding peptide, such as an antibody, arecompared. The antigen typically is selectively isotopically labeled with15N so that only signals corresponding to the antigen and no signalsfrom the antigen binding peptide are seen in the NMR-spectrum. Antigensignals originating from amino acids involved in the interaction withthe antigen binding peptide typically will shift position in thespectrum of the complex compared to the spectrum of the free antigen,and the amino acids involved in the binding can be identified that way.See, e. g., Ernst Schering Res Found Workshop. 2004; (44): 149-67; Huanget Journal of Molecular Biology, Vol. 281 (1) pp. 61-67 (1998); andSaito and Patterson, Methods. 1996 June; 9 (3): 516-24.

Epitope mapping/characterization also can be performed using massspectrometry methods. See, e.g., Downward, J Mass Spectrom. 2000 April;35 (4): 493-503 and Kiselar and Downard, Anal Chem. 1999 May 1; 71 (9):1792-801. Protease digestion techniques also can be useful in thecontext of epitope mapping and identification. Antigenicdeterminant-relevant regions/sequences can be determined by proteasedigestion, e.g. by using trypsin in a ratio of about 1:50 to TLR3 or o/ndigestion at and pH 7-8, followed by mass spectrometry (MS) analysis forpeptide identification. The peptides protected from trypsin cleavage bythe anti-TLR3 binder can subsequently be identified by comparison ofsamples subjected to trypsin digestion and samples incubated withantibody and then subjected to digestion by e.g. trypsin (therebyrevealing a footprint for the binder). Other enzymes like chymotrypsin,pepsin, etc., also or alternatively can be used in similar epitopecharacterization methods. Moreover, enzymatic digestion can provide aquick method for analyzing whether a potential antigenic determinantsequence is within a region of the TLR3 polypeptide that is not surfaceexposed and, accordingly, most likely not relevant in terms ofimmunogenicity/antigenicity. See, e. g., Manca, Ann Ist Super Sanita.1991; 27: 15-9 for a discussion of similar techniques.

Site-directed mutagenesis is another technique useful for elucidation ofa binding epitope. For example, in “alanine-scanning”, each residuewithin a protein segment is re-placed with an alanine residue, and theconsequences for binding affinity measured. If the mutation leads to asignificant resuction in binding affinity, it is most likely involved inbinding. Monoclonal antibodies specific for structural epitopes (i.e.,antibodies which do not bind the unfolded protein) can be used to verifythat the alanine-replacement does not influence over-all fold of theprotein. See, e.g., Clackson and Wells, Science 1995; 267:383-386; andWells, Proc Natl Acad Sci USA 1996; 93:1-6.

Electron microscopy can also be used for epitope “foot-printing”. Forexample, Wang et al., Nature 1992; 355:275-278 used coordinatedapplication of cryoelectron micros-copy, three-dimensional imagereconstruction, and X-ray crystallography to determine the physicalfootprint of a Fab-fragment on the capsid surface of native cowpeamosaic virus.

Other forms of “label-free” assay for epitope evaluation include surfaceplasmon resonance (SPR, BIACORE) and reflectometric interferencespectroscopy (RifS). See, e.g., Fägerstam et al., Journal Of MolecularRecognition 1990; 3:208-14; Nice et al., J. Chroma-togr. 1993;646:159-168; Leipert et al., Angew. Chem. Int. Ed. 1998; 37:3308-3311;Kroger et al., Biosensors and Bioelectronics 2002; 17:937-944.

It should also be noted that an antibody binding the same orsubstantially the same epitope as an antibody of the invention can beidentified in one or more of the exemplary competition assays describedherein.

Once antibodies are identified that are capable of binding TLR3 and/orhaving other desired properties, they will also typically be assessed,using standard methods including those described herein, for theirability to bind to other polypeptides, including unrelated polypeptidesand other TLR family members (e.g., human TLR1, 2, or 4-10). Ideally,the antibodies only bind with substantial affinity to TLR3, e.g., humanTLR3, and do not bind at a significant level to unrelated polypeptidesor to other TLR family members (e.g., TLR2 or TLR4; the amino acidsequence of human precursor TLR4 including a signal peptide at aminoacid residues 1-23 is found in NCBI accession number NP_612564, thedisclosure of which is incorporated herein by reference). However, itwill be appreciated that, as long as the affinity for TLR3 issubstantially greater (e.g., 5×, 10×, 50×, 100×, 500×, 1000×, 10,000×,or more) than it is for other TLR family members (or other, unrelatedpolypeptides), then the antibodies are suitable for use in the presentmethods.

The binding of the antibodies to TLR3-expressing cells can also beassessed in non-human primates, e.g. cynomolgus monkeys, or othermammals such as mice. The invention therefore provides an antibody, aswell as fragments and derivatives thereof, wherein said antibody,fragment or derivative specifically bind TLR3, and which furthermorebind TLR3 from non-human primates, e.g., cynomolgus monkeys. Optionally,cellular uptake or localization, optionally localization in asubcellular compartment such as the endocytic pathway, is assessed inorder to select an antibody that is readily taken up into the celland/or into the cellular compartment where it TLR3 is expressed.Cellular uptake or localization will generally be measured in the cellsin which the antibody is sought or believed to exert its activity, suchas in DC. Cellular uptake or localization can be assessed by standardmethods, such as by confocal staining using an antibody marked with adetectable moiety (e.g. a fluorescent moiety).

Upon immunization and production of antibodies in a vertebrate or cell,particular selection steps may be performed to isolate antibodies asclaimed. In this regard, in a specific embodiment, the invention alsorelates to methods of producing such antibodies, comprising: (a)immunizing a non-human mammal with an immunogen comprising a TLR3polypeptide; and (b) preparing antibodies from said immunized animal;and (c) selecting antibodies from step (b) that are capable of bindingTLR3. The antibodies can be tested for binding to TLR3 under acidicconditions corresponding to those in cytosolic compartments (e.g. theendosomic compartments), such as at a pH of between about 5.5 to 6.5.

The antibodies' bivalent binding affinity for human TLR3 under acidicconditions can determined Antibodies can be characterized for example bya mean K_(D) of no more than about (i.e. better affinity than) 100, 60,10, 5, or 1 nanomolar, preferably sub-nanomolar or optionally no morethan about 300, 200, 100 or 10 picomolar. K_(D) can be determined forexample for example by immobilizing recombinantly produced human TLR3proteins on a chip surface, followed by application of the antibody tobe tested in solution, e.g. as shown in the present Examples. To selectantibodies that retain binding similar binding under acidic and neutralconditions, one can seek to minimize the difference observed betweenbinding at neutral pH (e.g. 7.2) and acidic pH (e.g. a pH in the rangeof 4.5-6-5), for example where binding affinity at acidic pH is notsubstantially lower, e.g. where the K_(D) for binding to TLR3 decreasesby no more than 0.2-, 0.3-, 0.5-, 1.0-, or 1.5-log_(in), than thatobserved at non-acid pH. In one embodiment, the method further comprisesa step (d), selecting antibodies from (b) that are capable of competingfor binding to TLR3 with antibody 31C3, 29H3, 23C8, 28F11 or 34A3.

In one aspect of any of the embodiments, the antibodies preparedaccording to the present methods are monoclonal antibodies. In anotheraspect, the non-human animal used to produce antibodies according to themethods of the invention is a mammal, such as a rodent, bovine, porcine,fowl, horse, rabbit, goat, or sheep. The antibodies of the presentinvention encompass 31C3, 29H3, 23C8, 28F11 or 34A3. However, it will beappreciated that other antibodies can be obtained using the methodsdescribed herein, and thus antibodies of the invention can be antibodiesother than 31C3, 29H3, 23C8, 28F11 or 34A3. Additionally, antibodies ofthe invention can optionally be specified to be antibodies other thanany of antibodies TLR3.7 (eBioScience Inc., San Diego), antibody C1068of WO 06/060513, antibody C1130 of WO 2007/051164, any of the antibodiesdisclosed WO2010/051470, e.g., antibodies 1-19 and F17-F19, antibody40C1285 (Abcam), or antibodies 619F7, 713E4, 716G10, IMG-5631, IMG-315or IMG-5348 (all from Imgenex. Corp.) or derivatives of the foregoing,e.g. that comprise the antigen binding region in whole or in part.

According to an alternate embodiment, the DNA encoding an antibody thatbinds an epitope present on TLR3 polypeptides is isolated from thehybridoma of this invention and placed in an appropriate expressionvector for transfection into an appropriate host. The host is then usedfor the recombinant production of the antibody, or variants thereof,such as a humanized version of that monoclonal antibody, activefragments of the antibody, chimeric antibodies comprising the antigenrecognition portion of the antibody, or versions comprising a detectablemoiety.

DNA encoding the monoclonal antibodies of the invention, e.g., antibody31C3, 29H3, 23C8, 28F11 or 34A3, can be readily isolated and sequencedusing conventional procedures (e. g., by using oligonucleotide probesthat are capable of binding specifically to genes encoding the heavy andlight chains of murine antibodies). Once isolated, the DNA can be placedinto expression vectors, which are then transfected into host cells suchas E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells,or myeloma cells that do not otherwise produce immunoglobulin protein,to obtain the synthesis of monoclonal antibodies in the recombinant hostcells. As described elsewhere in the present specification, such DNAsequences can be modified for any of a large number of purposes, e.g.,for humanizing antibodies, producing fragments or derivatives, or formodifying the sequence of the antibody, e.g., in the antigen bindingsite in order to optimize the binding specificity of the antibody.

Recombinant expression in bacteria of DNA encoding the antibody is wellknown in the art (see, for example, Skerra et al., Curr. Opinion inImmunol., 5, pp. 256 (1993); and Pluckthun, Immunol. 130, p. 151 (1992).

Assessing the Ability of Antibodies to Modulate TLR3 Signaling

In certain embodiments, the antibodies of this invention are able tomodulate, e.g., inhibit signaling by, TLR3 polypeptides, andconsequently to modulate the activity or behavior of TLR3-expressingcells. For example, antibodies may inhibit the activation ofTLR3-expressing cells, e.g. they can inhibit the TLR3 signaling pathway,optionally without blocking the binding to TLR3 of natural or endogenousligands such as dsRNA; optionally they may block the ability of TLR3protein to form homodimers in the presence of a TLR3 ligand, thusblocking the initiation a signaling cascade. These antibodies are thusreferred to as “neutralizing” or “inhibitory” or “blocking” antibodies.Such antibodies are useful, inter alia, for decreasing the activity ofTLR3-expressing immune cells, e.g. for the treatment or prevention ofconditions involving excess TLR3-expressing cell activity or number, orwhere decreased TLR3-expressing cell activity can ameliorate, prevent,eliminate, or in any way improve the condition or any symptom thereof.

A range of cellular assays can be used to assess the ability of theantibodies to modulate TLR3 signaling. Any of a large number of assays,including molecular, cell-based, and animal-based models can be used toassess the ability of anti-TLR3 antibodies to modulate TLR3-expressingcell activity. For example, cell-based assays can be used in which cellsexpressing TLR3 are exposed to dsRNA, viral dsRNA, polyIC, or poly AU,or another TLR3 ligand and the ability of the antibody to disrupt thebinding of the ligand or the stimulation of the receptor (as determined,e.g., by examining any of the TLR3 cell activities addressed herein,such as interferon expression, NFkB activity, NK cell activation, etc.)is assessed. The TLR3 ligand used in the assays may be in any suitableform, including but not limited to as a purified ligand composition, ina mixture with non-TLR3 ligands, in a naturally occurring composition,in a cell or on the surface of a cell, or secreted by a cell (e.g. acell that produces ligand is used in the assay), in solution or on asolid support.

The activity of TLR3-expressing cells can also be assessed in theabsence of a ligand, by exposing the cells to the antibody itself andassessing its effect on any aspect of the cells' activity or behavior.In such assays, a baseline level of activity (e.g., cytokine production,proliferation, see below) of the TLR3-expressing cells is obtained inthe absence of a ligand, and the ability of the antibody or compound toalter the baseline activity level is detected. In one such embodiment, ahigh-throughput screening approach is used to identify compounds capableof affecting the activation of the receptor.

Any suitable physiological change that reflects TLR3 activity can beused to evaluate test antibodies or antibody derivatives. For example,one can measure a variety of effects, such as changes in gene expression(e.g., NFkB-responding genes), protein secretion (e.g., interferon),cell growth, cell proliferation, pH, intracellular second messengers,e.g., Ca²⁺, IP3, cGMP, or cAMP, or activity such as ability to activateNK cells. In one embodiment, the activity of the receptor is assessed bydetecting production of cytokines, e.g. TLR3-responsive cytokines,proinflammatory cytokines.

TLR3 modulation can be assessed using any of a number of possiblereadout systems, most based upon a TLR/IL-1R signal transductionpathway, involving, e.g., the MyD88-independent/TRIF dependent signaltransduction pathway, involving, e.g., IRF3, IRF7, IKKE and/or TBK1(Akira and Takeda (2004) Nature Review Immunol. 4:499-511). Thesepathways activate kinases including KB kinase complex. TLR3 activationcan be assessed by examining any aspect of TLR signaling. For example,activation of TLR signaling triggers alterations in protein-proteinassociations (e.g., TRIF with TBK and/or IKKE), in intracellularlocalization of proteins (such as movement of NK-kB into the nucleus),and in gene expression (e.g., in expression of NK-kB sensitive genes),and cytokine production (e.g., production and secretion of IFN-gamma,IL-6, IP10, MCP-1). Any such alteration can be detected and used todetect TLR3 activation. In one embodiment, TLR3 stimulation is detectedby collecting supernatants after 18-20 hr of culture and measuringlevels of IFN-gamma, IL-6, IP-10 and/or MCP-1 by sandwich ELISA. Inanother embodiment, TLR3 stimulation is detected by collectingsupernatants after 18-20 hr of culture and measuring levels ofIFN-gamma, IL-6, IP-10 and/or MCP-1 by sandwich ELISA.

In one embodiment, cells that naturally express TLR3 are used, such asDC (e.g. myeloid DC or monocyte derived DC. In another embodiment, cellsare used that contain a reporter construct that causes the expression ofa detectable gene product upon TLR3 stimulation and consequentactivation of the signal transduction pathway. Reporter genes andreporter gene constructs particularly useful for the assays include,e.g., a reporter gene operatively linked to a promoter sensitive toNF-kB or to signaling mediated by, particularly TRIF, IRF3, IRF7, IKKE,TBK1. Examples of such promoters include, without limitation, those forIL-1alpha, IL-6, IL-8, IL-12 p40, IP-10, CD80, CD86, and TNF-alpha. Thereporter gene operatively linked to the TLR-sensitive promoter caninclude, without limitation, an enzyme (e.g., luciferase, alkalinephosphatase, beta-galactosidase, chloramphenicol acetyltransferase(CAT), etc.), a bioluminescence marker (e.g., green-fluorescent protein(GFP, e.g., U.S. Pat. No. 5,491,084), blue fluorescent protein (BFP,e.g., U.S. Pat. No. 6,486,382), etc.), a surface-expressed molecule(e.g., CD25, CD80, CD86), and a secreted molecule (e.g., IL-1, IL-6,IL-8, IL-12 p40, TNF-alpha). See, e.g., Hacker H et al. (1999) EMBO J.18:6973-82; Murphy T L et al. (1995) Mol Cell Biol 15:5258-67, thedisclosures of which are herein incorporated by reference. Reporterplasmids suitable for use are commercially available (InvivoGen, SanDiego, Calif.). In one embodiment, the assay includes determining, in ahost cell made to express a human TLR3 polypeptide, whether a testcomposition induces luciferase expression (or other reporter) under thecontrol of a promoter responsive to TLR3 signaling (e.g. ISRE,IFN-stimulated response element).

In assays relying on enzyme activity readout, substrate can be suppliedas part of the assay, and detection can involve measurement ofchemoluminescence, fluorescence, color development, incorporation ofradioactive label, drug resistance, optical density, or other marker ofenzyme activity. For assays relying on surface expression of a molecule,detection can be accomplished using flow cytometry (FACS) analysis orfunctional assays. Secreted molecules can be assayed using enzyme-linkedimmunosorbent assay (ELISA) or bioassays. Many of these and othersuitable readout systems are well known in the art and are commerciallyavailable. Preferably, the reporter system, whichever used, isquantifiable.

In another embodiment, the effect of the antibodies on TLR3-expressingcells is assessed in non-human primates in vivo. For example, apharmaceutical composition comprising an anti-TLR3 antibody of thepresent invention is administered to a non-human primate that is eitherhealthy or affected by a condition, e.g. an autoimmune disease orinflammation and the effect of the administration on, e.g., the numberor activity of TLR3-expressing cells in the primate, the presence and/orlevels of cytokines, or on the progression of the condition is assessed.Any antibody or antibody derivative or fragment that effects adetectable change in any of these TLR3-related parameters is a candidatefor use in the herein-described methods.

In any of the herein-described assays, an increase or decrease of 5%,10%, 20%, preferably 30%, 40%, 50%, most preferably 60%, 70%, 80%, 90%,95%, or greater in any detectable measure of TLR3-stimulated activity inthe cells indicates that the test antibody is suitable for use in thepresent methods.

When assessing inhibitory anti-TLR3 antibodies, the antibodies can beadvantageously selected to modify any parameter associated withinflammation or autoimmunity. For example, antibodies can be selected toreduce activation, particularly production of pro-inflammatorycytokines, in cells. The cells may be, for example, cells obtained froman individual suffering from an inflammatory or autoimmune disorder.

Antibody CDR Sequences

In one aspect of any of the embodiments of the invention, an antibodymay comprise a heavy and/or light chain having CDR1, 2 and/or 3sequences according to the respective formula selected from Formulas (I)to (XX). In any embodiment herein, a particular HCDR1-3 or LCDR-1-3 maybe specified as having a sequence of Formulas (I) to (XX). In onepreferred embodiment, the antibody comprises a light chain comprisingthe three LCDRs and a heavy chain comprising the three HCDRs.Optionally, provided is an antibody where any of the light and/or heavychain variable regions are fused to an immunoglobulin constant region ofthe IgG type, optionally a human constant region, optionally an IgG1 orIgG4 isotype.

In one embodiment, LCDR1 is of Formula (I):

(I) (SEQ ID NO: 61) R-A-S-E-N-I-Y-S-Xaa₁-L-A,wherein Xaa₁ may be a conservative or non conservative substitution or adeletion or insertion, preferably, wherein Xaa₁ may be Ser, Tyr or Asn.

In one embodiment, LCDR2 is of Formula (II):

(II) (SEQ ID NO: 62) Xaa₂-A-K-T-L-A-E,wherein Xaa₂ may be a conservative or non conservative substitution or adeletion or insertion, preferably, wherein Xaa₂ may be Asn or Tyr.

In one embodiment, LCDR3 is of Formula (III):

(III) (SEQ ID NO: 63) Q-H-H-Y-G-T-P-Xaa₃-T,wherein Xaa₃ may be a conservative or non conservative substitution or adeletion or insertion, preferably, wherein Xaa₃ may be Tyr, Phe, Pro.

In one embodiment, LCDR1 is of Formula (IV):

(IV) (SEQ ID NO: 64) Xaa₄-A-S-Xaa₅-Xaa₆-Xaa₇-Xaa₈-Xaa₉-Xaa₁₀-Xaa₁₁-Xaa₁₂,wherein Xaa₄ to Xaa₁₂ may be a conservative or non conservativesubstitution or a deletion or insertion, preferably, wherein Xaa₄ may beArg, Ser or Lys, and/or Xaa₅ may be Glu, Ser or Gln, and/or Xaa₆ may beAsn or Ser, and/or Xaa₇ may be Ile or Val, and/or Xaa₈ may be adeletion, Tyr or Arg, and/or Xaa₉ may be Ser or Thr, and/or Xaa₁₀ may beTyr, Asn or Ser, and/or Xaa₁₁ may be Leu, Met or Val, and/or Xaa₁₂ maybe Ala or Phe.

In one embodiment, LCDR1 is of Formula (V):

(V) (SEQ ID NO: 65) Xaa₁₃-Xaa₁₄-Xaa₁₅-Xaa₁₆-L-A-Xaa₁₇wherein Xaa₁₃ to Xaa₁₇ may be a conservative or non conservativesubstitution or a deletion or insertion, preferably, wherein Xaa₁₃ maybe Leu, Asn or Tyr, and/or Xaa₁₄ may be Ala or Thr, and/or Xaa₁₅ may beLys or Ser, and/or Xaa₁₆ may be Asn or Thr, and/or Xaa₁₇ may be Glu orSer.

In one embodiment, LCDR2 is of Formula (VI):

(VI) (SEQ ID NO: 66) Xaa₁₈-A-Xaa₁₉-Xaa₂₀-Xaa₂₁-Xaa₂₂-Xaa₂₃wherein Xaa₁₉ to Xaa₂₃ may be a conservative or non conservativesubstitution or a deletion or insertion, preferably, wherein Xaa₁₈ maybe Tyr, Asn or Leu, and/or Xaa₁₉ may be Ser or Lys, and/or Xaa₂₀ may beAsn or Thr, and/or Xaa₂₁ may be Leu or Arg, and/or Xaa₂₂ may be Ala orHis, and/or Xaa₂₃ may be Thr or Glu.

In one embodiment, LCDR2 is of Formula (VII):

(VII) (SEQ ID NO: 67) L-Xaa₂₄-S-N-Xaa₂₅-Xaa₂₆-Xaa₂₇wherein Xaa₂₄ to Xaa₂₇ may be a conservative or non conservativesubstitution or a deletion or insertion, preferably, wherein Xaa₂₄ maybe Thr or Ala, and/or Xaa₂₅ may be Leu or Arg, and/or Xaa₂₆ may be Alaor His, and/or Xaa₂₇ may be Ser or Thr.

In one embodiment, LCDR3 is of Formula (VIII):

(VIII) (SEQ ID NO: 68) Q-Xaa₂₈-Xaa₂₉-Xaa₃₀-G-Xaa₃₁-P-Xaa₃₂-Twherein Xaa₂₈ to Xaa₃₂ may be a conservative or non conservativesubstitution or a deletion or insertion, preferably, wherein Xaa₂₈ maybe His or Gln, and/or Xaa₂₉ may be His or Trp, and/or Xaa₃₀ may be Tyror Thr, and/or Xaa₃₁ may be Thr or Asn, and/or Xaa₃₂ may be Tyr, Phe orPro.

In one embodiment, LCDR3 is of Formula (IX):

(IX) (SEQ ID NO: 69) Xaa₃₃-Xaa₃₄-H-Xaa₃₅-Xaa₃₆-Xaa₃₇-P-Xaa₃₈-Twherein Xaa₃₃ to Xaa₃₈ may be a conservative or non conservativesubstitution or a deletion or insertion, preferably, wherein Xaa₃₃ maybe Gln or Leu, and/or Xaa₃₄ may be His or Gln, and/or Xaa₃₅ may be Trpor Tyr, and/or Xaa₃₆ may be Asn or Gly, and/or Xaa₃₇ may be Tyr or Thr,and/or Xaa₃₈ may be Tyr, Phe or Pro.

In one embodiment, LCDR3 is of Formula (X):

(X) (SEQ ID NO: 70) Xaa₃₉-Q-Xaa₄₀-Xaa₄₁-Xaa₄₂-Xaa₄₃-P-Xaa₄₄-Twherein Xaa₄₀ to Xaa₄₄ may be a conservative or non conservativesubstitution or a deletion or insertion, preferably, wherein Xaa₃₉ maybe Gln or Leu, and/or Xaa₄₀ may be Trp or His, and/or Xaa₄₁ may be Thror Trp, and/or Xaa₄₂ may be Gly or Asn, and/or Xaa₄₃ may be Asn or Tyr,and/or Xaa₄₄ may be Pro or Tyr.

In one embodiment, HCDR1 is of Formula (XI):

(XI) (SEQ ID NO: 71) G-Y-S-F-T-G-Y-Xaa₄₅-Xaa₄₆-Hwherein Xaa₄₅ to Xaa₄₆ may be a conservative or non conservativesubstitution or a deletion or insertion, preferably, wherein Xaa₄₅ maybe Phe or Tyr, and/or Xaa₄₆ may be Met or Ile.

In one embodiment, HCDR1 is of Formula (XII):

(XII) (SEQ ID NO: 72) G-Y-S-F-T-Xaa₄₇-Y-Xaa₄₈-M-Hwherein Xaa₄₇ to Xaa₄₈ may be a conservative or non conservativesubstitution or a deletion or insertion, preferably, wherein Xaa₄₇ maybe Gly or Ala, and/or Xaa₄₈ may be Phe or Tyr.

In one embodiment, HCDR1 is of Formula (XIII):

(XIII) (SEQ ID NO: 73) G-Y-S-F-T-Xaa₄₉-Y-Y-Xaa₅₀-Hwherein Xaa₄₉ to Xaa₅₀ may be a conservative or non conservativesubstitution or a deletion or insertion, preferably, wherein Xaa₄₉ maybe Gly or Ala, and/or Xaa₅₀ may be Ile or Met.

In one embodiment, HCDR1 is of Formula (XIV):

(XIV) (SEQ ID NO: 74) G-Y-Xaa₅₁-F-T-Xaa₅₂-Y-Xaa₅₃-Xaa₅₄-Xaa₅₅wherein Xaa₅₁ to Xaa₅₅ may be a conservative or non conservativesubstitution or a deletion or insertion, preferably, wherein Xaa₅₁ maybe Val or Ser, and/or Xaa₅₂ may be Thr, Gly or Ala, and/or Xaa₅₃ may beSer, Tyr or Phe, and/or Xaa₅₄ may be Ile or Met, and/or Xaa₅₅ may be Tyror His.

In one embodiment, HCDR1 is of Formula (XV):

(XV) (SEQ ID NO: 75) G-Y-S-Xaa₅₆-T-Xaa₅₇-G-Y-Xaa₅₈-Xaa₅₉-Hwherein Xaa₅₆ to Xaa₅₉ may be a conservative or non conservativesubstitution or a deletion or insertion, preferably, wherein Xaa₅₆ maybe Ile or Phe, and/or Xaa₅₇ may be a deletion or Ser, and/or Xaa₅₈ maybe Ser, Tyr or Phe, and/or Xaa₅₉ may be Trp, Ile or Met.

In one embodiment, HCDR1 is of Formula (XVI):

(XVI) (SEQ ID NO: 76) G-Y-Xaa₆₀-Xaa₆₁-T-Xaa₆₂-Xaa₆₃-Y-S-Xaa₆₄-Xaa₆₅wherein Xaa₆₀ to Xaa₆₅ may be a conservative or non conservativesubstitution or a deletion or insertion, preferably, wherein Xaa₆₀ maybe Val or Ser and/or Xaa₆₁ may be Phe or Ile and/or Xaa₆₂ may be Thr orSer and/or Xaa₆₃ may be deletion or Gly and/or Xaa₆₄ may be Ile or Trpand/or Xaa₆₅ may be Tyr or His.

In one embodiment, HCDR2 is of Formula (XVII):

(XVII) (SEQ ID NO: 77) R-I-N-P-Y-Xaa₆₆-G-A-T-S-Xaa₆₇-N-Xaa₆₈-N-F-K-Dwherein Xaa₆₆ to Xaa₆₈ may be a conservative or non conservativesubstitution or a deletion or insertion, preferably, wherein Xaa₆₆ maybe Asn or Tyr and/or Xaa₆₇ may be deletion or Tyr and/or Xaa₆₈ may beArg or Gln.

In one embodiment, HCDR2 is of Formula (XVIII):

(XVIII) (SEQ ID NO: 78) R-I-N-P-Y-Xaa₆₆-G-A-T-S-Y-N-Q-N-F-K-Dwherein Xaa₆₆ may be a conservative or non conservative substitution ora deletion or insertion, preferably, wherein Xaa₆₆ may be Asn or Tyr

In one embodiment, HCDR2 is of Formula (XIX):

(XIX) (SEQ ID NO: 79) R-I-N-P-Y-N-G-A-T-S-Y-N-Xaa₆₈-N-F-K-Dwherein Xaa₆₈ may be a conservative or non conservative substitution ora deletion or insertion, preferably, wherein Xaa₆₈ may be Arg or Gln.

In one embodiment, HCDR2 is of Formula (XX):

(XX) (SEQ ID NO: 80) Y-I-Xaa₆₉-Xaa₇₀-Y-Xaa₇₁-G-Xaa₇₂-T-Xaa₇₃-Y-N-Xaa₇₄-Xaa₇₅-Xaa₇₆-Xaa₇₇-Xaa₇₈wherein Xaa₆₉ to Xaa₇₈ may be a conservative or non conservativesubstitution or a deletion or insertion, preferably, wherein Xaa₆₉ maybe Asp or His and/or Xaa₇₀ may be a deletion or Pro and/or Xaa₇₁ may beSer or Asn and/or Xaa₇₂ may be Ile or Asp and/or Xaa₇₃ may be Ser or Asnand/or Xaa₇₄ may be Gln or Pro and/or Xaa₇₅ may be Lys or Ser and/orXaa₇₆ may be Phe or Leu and/or Xaa₇₇ may be Lys or Arg and/or Xaa₇₈ maybe Gly or Ser.

In one embodiment, HCDR2 is of Formula (XXI):

(XXI) (SEQ ID NO: 81) Xaa₇₉-I-Xaa₈₀-Xaa₈₁-Y-Xaa₈₂-G-Xaa₈₃-T-Xaa₈₄-Xaa₈₅-N-Xaa₈₆-Xaa₈₇-Xaa₈₈-Xaa₈₉-Xaa₉₀wherein Xaa₇₉ to Xaa₉₀ may be a conservative or non conservativesubstitution or a deletion or insertion, preferably, wherein Xaa₇₉ maybe Arg or Tyr and/or Xaa₈₀ may be Asn, Asp or His and/or Xaa₈₁ may be adeletion or Pro and/or Xaa₈₂ may be Tyr, Asn or Ser and/or Xaa₈₃ may beIle, Asp or Ala and/or Xaa₈₄ may be Ser or Asn and/or Xaa₈₅ may be adeletion or Tyr and/or Xaa₈₆ may be Pro, Arg or Gln and/or Xaa₈₇ may beSer, Lys or Asn and/or Xaa₈₈ may be Phe or Leu and/or Xaa₈₉ may be Lysor Arg and/or Xaa₉₀ may be Asp or Gly.

In one embodiment, HCDR3 is of Formula (XXII):

(XXII) (SEQ ID NO: 82) Xaa₉₁-Xaa₉₂-G-Xaa₉₃-Xaa₉₄-Y-Xaa₉₅-F-D-Ywherein Xaa₉₁ to Xaa₉₅ may be a conservative or non conservativesubstitution or a deletion or insertion, preferably, wherein Xaa₉₁ maybe Asp or Ser, and/or Xaa₉₂ may be Asp or Gly, and/or Xaa₉₃ may be Glyor Asn, and/or Xaa₉₄ may be Asn or Thr, and/or Xaa₉₅ may be Pro or adeletion.

In one embodiment, HCDR3 is of Formula (XXIII):

(XXIII) (SEQ ID NO: 83)Xaa₉₆-Xaa₉₇-Xaa₉₈-Xaa₉₉-Xaa₁₀₀-Y-Xaa₁₀₁-Xaa₁₀₂-D-Ywherein Xaa₉₆ to Xaa₁₀₂ may be a conservative or non conservativesubstitution or a deletion or insertion, preferably, wherein Xaa₉₆ maybe S or D, and/or Xaa₉₇ may be G, T D, and/or Xaa₉₈ may be G, K or adeletion, and/or Xaa₉₉ may be G, L, N or a deletion, and/or Xaa₁₀₀ maybe Y, T, G, N, and/or Xaa₁₀₁ may be P, G or a deletion, and/or Xaa₁₀₂may be M, F or L.

In one embodiment, HCDR3 is of Formula (XXIV):

(XXIV) (SEQ ID NO: 84) Xaa₁₀₃-Xaa₁₀₄-Xaa₁₀₅-Xaa₁₀₆-Xaa₁₀₇-Xaa₁₀₈-Xaa₁₀₉-F-D-Ywherein Xaa₁₀₃ to Xaa₁₀₉ may be a conservative or non conservativesubstitution or a deletion or insertion, preferably, wherein Xaa₁₀₃ maybe Asp, Ser, Glu, and/or Xaa₁₀₄ may be Asp or Gly, and/or Xaa₁₀₅ may beGly or Asn, and/or Xaa₁₀₆ may be Asn, Tyr or Gly, and/or Xaa₁₀₇ may beAsn, Thr or Tyr, and/or Xaa₁₀₈ may be Tyr or Gly, and/or Xaa₁₀₉ may beTyr, Pro or a deletion

In one embodiment, HCDR3 is of Formula (XXV):

(XXV) (SEQ ID NO: 85) Xaa₁₁₀-G-Xaa₁₁₁-Xaa₁₁₂-Y-Xaa₁₁₃-Xaa₁₁₄-Xaa₁₁₅-D-Ywherein Xaa₁₁₀ to Xaa₁₁₅ may be a conservative or non conservativesubstitution or a deletion or insertion, preferably, wherein Xaa₁₁₀ maybe Glu or Asp, and/or Xaa₁₁₁ may be Asn or a deletion, and/or Xaa₁₁₂ maybe Tyr or a deletion, and/or Xaa₁₁₃ may be Tyr, or Gly, and/or Xaa₁₁₄may be Tyr or Gly, and/or Xaa₁₁₅ may be Met or Phe.

In one embodiment, an antibody of the invention may comprise a lightchain comprising:

-   -   a a light chain CDR1 (LCDR1) amino acid sequence selected from        SEQ ID NOS: 61, 64 and 65; and/or    -   b a light chain CDR2 (LCDR2) amino acid sequence selected from        SEQ ID NOS: 62, 66 and 67; and/or    -   c a light chain CDR3 (LCDR3) amino acid sequence selected from        SEQ ID NOS: 63, 68, 69 and 70.

In one embodiment, an antibody of the invention may comprise a heavychain comprising:

-   -   a a heavy chain CDR1 (HCDR1) amino acid sequence selected from        SEQ ID NOS: 71 to 76; and/or    -   b a heavy chain CDR2 (HCDR2) amino acid sequence selected from        SEQ ID NOS: 77 to 81; and/or    -   c a heavy chain CDR3 (HCDR3) amino acid sequence selected from        SEQ ID NOS: 82 to 85.

Antibody 29H3

Cells producing antibody 29H3 have been deposited at the CNCM underaccession number I-4187; the antibody 29H3 has also been sequenced. Theamino acid sequence of the heavy chain variable region is listed as SEQID NO:10, the amino acid sequence of the light chain variable region islisted as SEQ ID NO:11. The nucleic acid sequence encoding the heavy andlight chain variable regions are listed in SEQ ID NOS 53 and 54,respectively. In one embodiment, the invention provides an antibody thatbinds essentially the same epitope or determinant as monoclonalantibodies 29H3; optionally the antibody comprises an antigen bindingregion of antibody 29H3. In any of the embodiments herein, antibody 29H3can be characterized by its amino acid sequence and/or nucleic acidsequence encoding it. In one preferred embodiment, the monoclonalantibody comprises the Fab or F(ab′)₂ portion of 29H3. Also provided isa monoclonal antibody that comprises the heavy chain variable region of29H3. According to one embodiment, the monoclonal antibody comprises thethree CDRs of the heavy chain variable region of 29H3. Also provided isa monoclonal antibody that further comprises the variable light chainvariable region of 29H3 or one, two or three of the CDRs of the lightchain variable region of 29H3. Optionally any one or more of said lightor heavy chain CDRs may contain one, two, three, four or five amino acidmodifications (e.g. substitutions, insertions or deletions). Optionally,provided is an antibody where any of the light and/or heavy chainvariable regions comprising part or all of an antigen binding region ofantibody 29H3 are fused to an immunoglobulin constant region of the IgGtype, optionally a human constant region, optionally an IgG1 or IgG4isotype. In another preferred embodiment the antibody is 29H3.

In another aspect, the invention provides a purified polypeptide whichencodes an antibody, wherein the antibody comprises: a VHCDR1 regioncomprising an amino acid sequence as set forth in SEQ ID NO:12, whereinone or more of these amino acids may be substituted by a different aminoacid; a VHCDR2 region comprising an amino acid sequence as set forth inSEQ ID NO:13, wherein one or more of these amino acids may besubstituted by a different amino acid; a VHCDR3 region comprising anamino acid sequence as set forth in SEQ ID NO:14, wherein one or more ofthese amino acids may be substituted by a different amino acid; a VLCDR1region comprising an amino acid sequence as set forth in SEQ ID NO:15,wherein one or more of these amino acids may be substituted by adifferent amino acid; a VLCDR2 region comprising an amino acid sequenceas set forth in SEQ ID NO:16, wherein one or more of these amino acidsmay be substituted by a different amino acid; and/or a VLCDR3 regioncomprising an amino acid sequence as set forth in SEQ ID NO:17, whereinone or more of these amino acids may be substituted by a different aminoacid.

In still another aspect, the invention provides an antibody, whichcomprises a heavy chain and/or a light chain each having at least threeCDRs, wherein one, two or three of at least three CDRs has the sequenceof SEQ ID NO:12 to 14 and 15 to 17 for the respective the heavy andlight chains, and which antibody specifically binds to TLR3 in acidicconditions.

In another aspect, the invention provides an antibody that binds humanTLR3, comprising:

-   -   a the heavy chain variable region of SEQ ID NO:10, wherein one,        two, three or more of these amino acids may be substituted by a        different amino acid; or    -   b the light chain variable region of SEQ ID NO:11, wherein one,        two, three or more of these amino acids may be substituted by a        different amino acid; or    -   c the heavy chain variable region of SEQ ID NO:10, wherein one,        two, three or more of these amino acids may be substituted by a        different amino acid; and the light chain variable region of SEQ        ID NO:11, wherein one or more of these amino acids may be        substituted by a different amino acid; or    -   d the heavy chain CDR 1, 2 and 3 (HCDR1, HCDR2, HCDR3) amino        acid sequences as shown in SEQ ID NO:12, 13 and 14, wherein one,        two, three or more of these amino acids may be substituted by a        different amino acid; or    -   e the light chain CDR 1, 2 and 3 (LCDR1, LCDR2, LCDR3) e amino        acid sequences as shown in SEQ ID NO:15, 16 and 17,        respectively, wherein one, two, three or more of these amino        acids may be substituted by a different amino acid; or    -   f the heavy chain CDR 1, 2 and 3 (HCDR1, HCDR2, HCDR3) amino        acid sequences as shown in SEQ ID NO: 12, 13 and 14, wherein        one, two, three or more of these amino acids may be substituted        by a different amino acid; and the light chain CDR 1, 2 and 3        (LCDR1, LCDR2, LCDR3) amino acid sequences as shown in SEQ ID        NO: 15, 16 and 17, wherein one, two, three or more of these        amino acids may be substituted by a different amino acid; or    -   g the heavy chain variable region which is at least 60%, 70%,        80%, 85%, 90% or 95% identical to the variable region having an        amino acid sequence of SEQ ID NO:10, wherein one, two, three or        more of these amino acids may be substituted by a different        amino acid; or    -   h the light chain variable region which is at least 60%, 70%,        80%, 85%, 90% or 95% identical to the variable region having an        amino acid sequence of SEQ ID NO:11, wherein one, two, three or        more of these amino acids may be substituted by a different        amino acid.

In another aspect of any of the embodiments herein, any of the CDRs 1, 2and 3 of the heavy and light chains may be characterized as having anamino acid sequence that shares at least 50%, 60%, 70%, 80%, 85%, 90% or95% sequence identity with the particular CDR or set of CDRs listed inthe corresponding SEQ ID NO.

In another aspect, the invention provides an antibody that competes forTLR3 binding with a monoclonal antibody of (a) to (h), above.

Antibody 31C3

Cells producing antibody 31C3 have been deposited at the CNCM underaccession number I-4186, also the antibody 31C3 has also been sequenced.The amino acid sequence of the heavy chain variable region is listed asSEQ ID NO: 2, the amino acid sequence of the light chain variable regionis listed as SEQ ID NO: 3. The nucleic acid sequence encoding the heavyand light chain variable regions are listed in SEQ ID NOS 51 and 52,respectively. In a specific embodiment, the invention provides anantibody that binds essentially the same epitope or determinant asmonoclonal antibodies 31C3; optionally the antibody comprises an antigenbinding region of antibody 31C3. In any of the embodiments herein,antibody 31C3 can be characterized by its amino acid sequence and/ornucleic acid sequence encoding it. In one preferred embodiment, themonoclonal antibody comprises the Fab or F(ab′)₂ portion of 31C3. Alsoprovided is a monoclonal antibody that comprises the heavy chainvariable region of 31C3. According to one embodiment, the monoclonalantibody comprises the three CDRs of the heavy chain variable region of31C3. Also provided is a monoclonal antibody that further comprises thevariable light chain variable region of 31C3 or one, two or three of theCDRs of the light chain variable region of 31C3. Optionally any one ormore of said light or heavy chain CDRs may contain one, two, three, fouror five amino acid modifications (e.g. substitutions, insertions ordeletions). Optionally, provided is an antibody where any of the lightand/or heavy chain variable regions comprising part or all of an antigenbinding region of antibody 31C3 are fused to an immunoglobulin constantregion of the IgG type, optionally a human constant region, optionally ahuman IgG1 or IgG4 isotype. In another preferred embodiment the antibodyis 31C3.

In another aspect, the invention provides a purified polypeptide whichencodes a antibody, wherein the antibody comprises: a VHCDR1 regioncomprising an amino acid sequence as set forth in SEQ ID NO:4, whereinone or more of these amino acids may be substituted by a different aminoacid; a VHCDR2 region comprising an amino acid sequence as set forth inSEQ ID NO:5, wherein one or more of these amino acids may be substitutedby a different amino acid; a VHCDR3 region comprising an amino acidsequence as set forth in SEQ ID NO:6, wherein one or more of these aminoacids may be substituted by a different amino acid; a VLCDR1 regioncomprising an amino acid sequence as set forth in SEQ ID NO:7, whereinone or more of these amino acids may be substituted by a different aminoacid; a VLCDR2 region comprising an amino acid sequence as set forth inSEQ ID NO:8, wherein one or more of these amino acids may be substitutedby a different amino acid; a VLCDR3 region comprising an amino acidsequence as set forth in SEQ ID NO:9, wherein one or more of these aminoacids may be substituted by a different amino acid.

In still another aspect, the invention provides an antibody, whichcomprises a heavy chain and/or a light chain each having at least threeCDRs, wherein one, two or three of the at least three CDRs has thesequence of SEQ ID NO:4 to 6 and 7 to 9 for the respective the heavy andlight chains, and which antibody specifically binds to TLR3 in acidicconditions.

In another aspect, the invention provides an antibody that binds humanTLR3, comprising:

-   -   a the heavy chain variable region of SEQ ID NO:2, wherein one,        two, three or more of these amino acids may be substituted by a        different amino acid; or    -   b the light chain variable region of SEQ ID NO: 3, wherein one,        two, three or more of these amino acids may be substituted by a        different amino acid; or    -   c the heavy chain variable region of SEQ ID NO: 2, wherein one        or more of these amino acids may be substituted by a different        amino acid; and the light chain variable region of SEQ ID NO: 3,        wherein one, two, three or more of these amino acids may be        substituted by a different amino acid; or    -   d the heavy chain CDR 1 and 2 (HCDR1, HCDR2) amino acid        sequences as shown in SEQ ID NO:4 and 5, wherein one, two, three        or more of these amino acids may be substituted by a different        amino acid, optionally wherein the heavy chain comprises CDR 1,        2 and 3 (HCDR1, HCDR2, HCDR3) amino acid sequences as shown in        SEQ ID NO:4, 5 and 6, wherein one, two, three or more of these        amino acids may be substituted by a different amino acid; or    -   e the light chain CDR 1, 2 and 3 (LCDR1, LCDR2, LCDR3) amino        acid sequences as shown in SEQ ID NO: 7, 8 and 9, wherein one,        two, three or more of these amino acids may be substituted by a        different amino acid; or    -   f the heavy chain CDR 1, 2 and 3 (HCDR1, HCDR2, HCDR3) amino        acid sequences as shown in SEQ ID NO: 4, 5 and 6, wherein one or        more of these amino acids may be substituted by a different        amino acid; and the light chain CDRs 1, 2 and 3 (LCDR1, LCDR2,        LCDR3) amino acid sequences as shown in SEQ ID NO: 7, 8 and 9,        wherein one, two, three or more of these amino acids may be        substituted by a different amino acid; or    -   g the heavy chain variable region which is at least 60%, 70%,        80%, 85%, 90% or 95% identical to the variable region having an        amino acid sequence of SEQ ID NO: 2, wherein one, two, three or        more of these amino acids may be substituted by a different        amino acid; or    -   h the light chain variable region which is at least 60%, 70%,        80%, 85%, 90% or 95% identical to the variable region having an        amino acid sequence of SEQ ID NO: 3, wherein one, two, three or        more of these amino acids may be substituted by a different        amino acid.

In another aspect of any of the embodiments herein, any of the CDRs 1, 2and 3 of the heavy and light chains may be characterized as having anamino acid sequence that shares at least 50%, 60%, 70%, 80%, 85%, 90% or95% sequence identity with the particular CDR or set of CDRs listed inthe corresponding SEQ ID NO.

In another aspect, the invention provides an antibody that competes forTLR3 binding with a monoclonal antibody of (a) to (h), above.

Antibody 23C8

The antibody 23C8 has been sequenced. The amino acid sequence of theheavy chain variable region is listed as SEQ ID NO:26, the amino acidsequence of the light chain variable region is listed as SEQ ID NO:27.The nucleic acid sequence encoding the heavy and light chain variableregions are listed in SEQ ID NOS 57 and 58, respectively. In a specificembodiment, the invention provides an antibody that binds essentiallythe same epitope or determinant as monoclonal antibodies 23C8;optionally the antibody comprises an antigen binding region of antibody23C8. In any of the embodiments herein, antibody 23C8 can becharacterized by its amino acid sequence and/or nucleic acid sequenceencoding it. In one preferred embodiment, the monoclonal antibodycomprises the Fab or F(ab′)2 portion of 23C8. Also provided is amonoclonal antibody that comprises the heavy chain variable region of23C8. According to one embodiment, the monoclonal antibody comprises thethree CDRs of the heavy chain variable region of 23C8. Also provided isa monoclonal antibody that further comprises the variable light chainvariable region of 23C8 or one, two or three of the CDRs of the lightchain variable region of 23C8. Optionally any one or more of said lightor heavy chain CDRs may contain one, two, three, four or five amino acidmodifications (e.g. substitutions, insertions or deletions). Optionally,provided is an antibody where any of the light and/or heavy chainvariable regions comprising part or all of an antigen binding region ofantibody 23C8 are fused to an immunoglobulin constant region of the IgGtype, optionally a human constant region, optionally a human IgG1 orIgG4 isotype. In another preferred embodiment the antibody is 23C8.

In another aspect, the invention provides a purified polypeptide whichencodes an antibody, wherein the antibody comprises: a VHCDR1 regioncomprising an amino acid sequence as set forth in SEQ ID NO:28, whereinone or more of these amino acids may be substituted by a different aminoacid; a VHCDR2 region comprising an amino acid sequence as set forth inSEQ ID NO:29, wherein one or more of these amino acids may besubstituted by a different amino acid; a VHCDR3 region comprising anamino acid sequence as set forth in SEQ ID NO:30, wherein one or more ofthese amino acids may be substituted by a different amino acid; a VLCDR1region comprising an amino acid sequence as set forth in SEQ ID NO:31,wherein one or more of these amino acids may be substituted by adifferent amino acid; a VLCDR2 region comprising an amino acid sequenceas set forth in SEQ ID NO:32, wherein one or more of these amino acidsmay be substituted by a different amino acid; a VLCDR3 region comprisingan amino acid sequence as set forth in SEQ ID NO:33, wherein one or moreof these amino acids may be substituted by a different amino acid.

In still another aspect, the invention provides an antibody, whichcomprises a heavy chain and/or a light chain each having at least threeCDRs, wherein one, two or three of at least three CDRs has the sequenceof SEQ ID NO:28 to 30 and 31 to 33 for the respective the heavy andlight chains, and which antibody specifically binds to TLR3 in acidicconditions.

In another aspect, the invention provides an antibody that binds humanTLR3, comprising:

-   -   a the heavy chain variable region of SEQ ID NO: 26, wherein one,        two, three or more of these amino acids may be substituted by a        different amino acid; or    -   b the light chain variable region of SEQ ID NO: 27, wherein one,        two, three or more of these amino acids may be substituted by a        different amino acid; or    -   c the heavy chain variable region of SEQ ID NO: 26, wherein one,        two, three or more of these amino acids may be substituted by a        different amino acid; and the light chain variable region of SEQ        ID NO: 27, wherein one, two, three or more of these amino acids        may be substituted by a different amino acid; or    -   d the heavy chain CDR 1 and 2 (HCDR1, HCDR2) amino acid        sequences as shown in SEQ ID NO: 28 and 29, wherein one, two,        three or more of these amino acids may be substituted by a        different amino acid; optionally wherein the heavy chain        comprises the CDR 1, 2 and 3 (HCDR1, HCDR2, HCDR3) amino acid        sequences as shown in SEQ ID NO: 28, 29 and 30, wherein one,        two, three or more of these amino acids may be substituted by a        different amino acid; or    -   e the light chain CDR 1, 2 and 3 (LCDR1, LCDR2, LCDR3) amino        acid sequences as shown in SEQ ID NO: 31, 32 and 33, wherein        one, two, three or more of these amino acids may be substituted        by a different amino acid; or    -   f the heavy chain CDR 1, 2 and 3 (HCDR1, HCDR2, HCDR3) amino        acid sequences as shown in SEQ ID NO: 28, 29 and 30, wherein        one, two, three or more of these amino acids may be substituted        by a different amino acid; and the light chain CDR 1, 2 and 3        (LCDR1, LCDR2, LCDR3) amino acid sequences as shown in SEQ ID        NO: 31, 32 and 33, wherein one, two, three or more of these        amino acids may be substituted by a different amino acid; or    -   g the heavy chain variable region which is at least 60%, 70%,        80%, 85%, 90% or 95% identical to the variable region having an        amino acid sequence of SEQ ID NO: 26, wherein one, two, three or        more of these amino acids may be substituted by a different        amino acid; or    -   h the light chain variable region which is at least 60%, 70%,        80%, 85%, 90% or 95% identical to the variable region having an        amino acid sequence of SEQ ID NO: 27, wherein one, two, three or        more of these amino acids may be substituted by a different        amino acid.

In another aspect of any of the embodiments herein, any of the CDRs 1, 2and 3 of the heavy and light chains may be characterized as having anamino acid sequence that shares at least 50% 60%, 70%, 80%, 85%, 90% or95% sequence identity with the particular CDR or set of CDRs listed inthe corresponding SEQ ID NO.

In another aspect, the invention provides an antibody that competes forTLR3 binding with a monoclonal antibody of (a) to (h), above.

Antibody 28F11

The antibody 28F11 has been sequenced. The amino acid sequence of theheavy chain variable region is listed as SEQ ID NO:18, the amino acidsequence of the light chain variable region is listed as SEQ ID NO:19.The nucleic acid sequence encoding the heavy and light chain variableregions are listed in SEQ ID NOS 55 and 56, respectively. In a specificembodiment, the invention provides an antibody that binds essentiallythe same epitope or determinant as monoclonal antibodies 28F11;optionally the antibody comprises an antigen binding region of antibody28F11. In any of the embodiments herein, antibody 28F11 can becharacterized by its amino acid sequence and/or nucleic acid sequenceencoding it. In one preferred embodiment, the monoclonal antibodycomprises the Fab or F(ab′)2 portion of 28F11. Also provided is amonoclonal antibody that comprises the heavy chain variable region of28F11. According to one embodiment, the monoclonal antibody comprisesthe three CDRs of the heavy chain variable region of 28F11. Alsoprovided is a monoclonal antibody that further comprises the variablelight chain variable region of 28F11 or one, two or three of the CDRs ofthe light chain variable region of 28F11. Optionally any one or more ofsaid light or heavy chain CDRs may contain one, two, three, four or fiveamino acid modifications (e.g. substitutions, insertions or deletions).Optionally, provided is an antibody where any of the light and/or heavychain variable regions comprising part or all of an antigen bindingregion of antibody 28F11 are fused to an immunoglobulin constant regionof the IgG type, optionally a human constant region, optionally a humanIgG1 or IgG4 isotype. In another preferred embodiment the antibody is28F11.

In another aspect, the invention provides a purified polypeptide whichencodes a antibody, wherein the antibody comprises: a VHCDR1 regioncomprising an amino acid sequence as set forth in SEQ ID NO:20, whereinone or more of these amino acids may be substituted by a different aminoacid; a VHCDR2 region comprising an amino acid sequence as set forth inSEQ ID NO:21, wherein one or more of these amino acids may besubstituted by a different amino acid; a VHCDR3 region comprising anamino acid sequence as set forth in SEQ ID NO:22, wherein one or more ofthese amino acids may be substituted by a different amino acid; a VLCDR1region comprising an amino acid sequence as set forth in SEQ ID NO:23,wherein one or more of these amino acids may be substituted by adifferent amino acid; a VLCDR2 region comprising an amino acid sequenceas set forth in SEQ ID NO:24, wherein one or more of these amino acidsmay be substituted by a different amino acid; a VLCDR3 region comprisingan amino acid sequence as set forth in SEQ ID NO:25, wherein one or moreof these amino acids may be substituted by a different amino acid.

In still another aspect, the invention provides an antibody, whichcomprises a heavy chain and/or a light chain each having at least threeCDRs, wherein one, two or three of at least three CDRs has the sequenceof SEQ ID NO: 20 to 22 and 23 to 25 for the respective heavy and lightchains, and which antibody specifically binds to TLR3 in acidicconditions.

In another aspect, the invention provides an antibody that binds humanTLR3, comprising:

-   -   a the heavy chain variable region of SEQ ID NO: 18, wherein one,        two, three or more of these amino acids may be substituted by a        different amino acid; or    -   b the light chain variable region of SEQ ID NO: 19, wherein one,        two, three or more of these amino acids may be substituted by a        different amino acid; or    -   c the heavy chain variable region of SEQ ID NO: 18, wherein one,        two, three or more of these amino acids may be substituted by a        different amino acid; and the light chain variable region of SEQ        ID NO: 19, wherein one, two, three or more of these amino acids        may be substituted by a different amino acid; or    -   d the heavy chain CDR 1 and 2 (HCDR1, HCDR2) amino acid        sequences as shown in SEQ ID NO: 20 and 21, wherein one, two,        three or more of these amino acids may be substituted by a        different amino acid; optionally wherein heavy chain comprises        CDR 1, 2 and 3 (HCDR1, HCDR2, HCDR3) amino acid sequences as        shown in SEQ ID NO: 20, 21 and 22, wherein one, two, three or        more of these amino acids may be substituted by a different        amino acid; or    -   e the light chain CDR 1, 2 and 3 (LCDR1, LCDR2, LCDR3) amino        acid sequences as shown in SEQ ID NO: 23, 24 and 25, wherein        one, two, three or more of these amino acids may be substituted        by a different amino acid; or    -   f the heavy chain CDR 1, 2 and 3 (HCDR1, HCDR2, HCDR3) amino        acid sequences as shown in SEQ ID NO: 20, 21 and 22, wherein        one, two, three or more of these amino acids may be substituted        by a different amino acid; and the light chain CDR 1, 2 and 3        (LCDR1, LCDR2, LCDR3) amino acid sequences as shown in SEQ ID        NO: 23, 24 and 25, wherein one, two, three or more of these        amino acids may be substituted by a different amino acid; or    -   g the heavy chain variable region which is at least 60%, 70%,        80%, 85%, 90% or 95% identical to the variable region having an        amino acid sequence of SEQ ID NO: 18, wherein one, two, three or        more of these amino acids may be substituted by a different        amino acid; or    -   h the light chain variable region which is at least 60%, 70%,        80%, 85%, 90% or 95% identical to the variable region having an        amino acid sequence of SEQ ID NO: 19, wherein one, two, three or        more of these amino acids may be substituted by a different        amino acid.

In another aspect of any of the embodiments herein, any of the CDRs 1, 2and 3 of the heavy and light chains may be characterized as having anamino acid sequence that shares at least 50%, 60%, 70%, 80%, 85%, 90% or95% sequence identity with the particular CDR or set of CDRs listed inthe corresponding SEQ ID NO.

In another aspect, the invention provides an antibody that competes forTLR3 binding with a monoclonal antibody of (a) to (h), above.

Antibody 34A3

The antibody 34A3 has been sequenced. The amino acid sequence of theheavy chain variable region is listed as SEQ ID NO:34, the amino acidsequence of the light chain variable region is listed as SEQ ID NO:35.The nucleic acid sequence encoding the heavy and light chain variableregions are listed in SEQ ID NOS 59 and 60, respectively. In a specificembodiment, the invention provides an antibody that binds essentiallythe same epitope or determinant as monoclonal antibodies 34A3;optionally the antibody comprises an antigen binding region of antibody34A3. In any of the embodiments herein, antibody 34A3 can becharacterized by its amino acid sequence and/or nucleic acid sequenceencoding it. In one preferred embodiment, the monoclonal antibodycomprises the Fab or F(ab′)2 portion of 34A3. Also provided is amonoclonal antibody that comprises the heavy chain variable region of34A3. According to one embodiment, the monoclonal antibody comprises thethree CDRs of the heavy chain variable region of 34A3. Also provided isa monoclonal antibody that further comprises the variable light chainvariable region of 34A3 or one, two or three of the CDRs of the lightchain variable region of 34A3. Optionally any one or more of said lightor heavy chain CDRs may contain one, two, three, four or five amino acidmodifications (e.g. substitutions, insertions or deletions). Optionally,provided is an antibody where any of the light and/or heavy chainvariable regions comprising part or all of an antigen binding region ofantibody 34A3 are fused to an immunoglobulin constant region of the IgGtype, optionally a human constant region, optionally a human IgG1 orIgG4 isotype. In another preferred embodiment the antibody is 34A3.

In another aspect, the invention provides a purified polypeptide whichencodes a antibody, wherein the antibody comprises: a VHCDR1 regioncomprising an amino acid sequence as set forth in SEQ ID NO:36, whereinone or more of these amino acids may be substituted by a different aminoacid; a VHCDR2 region comprising an amino acid sequence as set forth inSEQ ID NO:37, wherein one or more of these amino acids may besubstituted by a different amino acid; a VHCDR3 region comprising anamino acid sequence as set forth in SEQ ID NO:38, wherein one or more ofthese amino acids may be substituted by a different amino acid; a VLCDR1region comprising an amino acid sequence as set forth in SEQ ID NO:39,wherein one or more of these amino acids may be substituted by adifferent amino acid; a VLCDR2 region comprising an amino acid sequenceas set forth in SEQ ID NO:40, wherein one or more of these amino acidsmay be substituted by a different amino acid; a VLCDR3 region comprisingan amino acid sequence as set forth in SEQ ID NO:41, wherein one or moreof these amino acids may be substituted by a different amino acid.

In still another aspect, the invention provides an antibody, whichcomprises a heavy chain and/or a light chain each having at least threeCDRs, wherein one, two or three of at least three CDRs has the sequenceof SEQ ID NO:36 to 38 and 29 to 41 for the respective heavy and lightchains, and which antibody specifically binds to TLR3 in acidicconditions.

In another aspect, the invention provides an antibody that binds humanTLR3, comprising:

-   -   a the heavy chain variable region of SEQ ID NO: 34, wherein one,        two, three or more of these amino acids may be substituted by a        different amino acid; or    -   b the light chain variable region of SEQ ID NO: 35, wherein one,        two, three or more of these amino acids may be substituted by a        different amino acid; or    -   c the heavy chain variable region of SEQ ID NO: 34, wherein one,        two, three or more of these amino acids may be substituted by a        different amino acid; and the light chain variable region of SEQ        ID NO: 35, wherein one, two, three or more of these amino acids        may be substituted by a different amino acid; or    -   d the heavy chain CDRs 1, 2 and 3 (HCDR1, HCDR2, HCDR3) amino        acid sequences as shown in

SEQ ID NO: 36, 37 and 38, wherein one, two, three or more of these aminoacids may be substituted by a different amino acid; or

-   -   e the light chain CDRs 1, 2 and 3 (LCDR1, LCDR2, LCDR3) amino        acid sequences as shown in SEQ ID NO: 39, 40 and 41, wherein one        or more of these amino acids may be substituted by a different        amino acid; or    -   f the heavy chain CDRs 1, 2 and 3 (HCDR1, HCDR2, HCDR3) amino        acid sequences as shown in SEQ ID NO: 36, 37 and 38, wherein        one, two, three or more of these amino acids may be substituted        by a different amino acid; and the light chain CDRs 1, 2 and 3        (LCDR1, LCDR2, LCDR3) amino acid sequences as shown in SEQ ID        NO: 39, 40 and 41, wherein one, two, three or more of these        amino acids may be substituted by a different amino acid; or    -   g the heavy chain variable region which is at least 60%, 70%,        80%, 85%, 90% or 95% identical to the variable region having an        amino acid sequence of SEQ ID NO: 34, wherein one, two, three or        more of these amino acids may be substituted by a different        amino acid; or    -   h the light chain variable region which is at least 60%, 70%,        80%, 85%, 90% or 95% identical to the variable region having an        amino acid sequence of SEQ ID NO: 35, wherein one, two, three or        more of these amino acids may be substituted by a different        amino acid.

In another aspect of any of the embodiments herein, any of the CDRs 1, 2and 3 of the heavy and light chains may be characterized as having anamino acid sequence that shares at least 50%, 60%, 70%, 80%, 85%, 90% or95% sequence identity with the particular CDR or set of CDRs listed inthe corresponding SEQ ID NO.

In another aspect, the invention provides an antibody that competes forTLR3 binding with a monoclonal antibody of (a) to (h), above.

In any of the antibodies of the invention, e.g, 31C3, 29H3, 23C8, 28F11or 34A3, the specified variable region and CDR sequences may compriseconservative sequence modifications. Conservative sequence modificationsrefers to amino acid modifications that do not significantly affect oralter the binding characteristics of the antibody containing the aminoacid sequence. Such conservative modifications include amino acidsubstitutions, additions and deletions. Modifications can be introducedinto an antibody of the invention by standard techniques known in theart, such as site-directed mutagenesis and PCR-mediated mutagenesis.Conservative amino acid substitutions are typically those in which anamino acid residue is replaced with an amino acid residue having a sidechain with similar physicochemical properties. Specified variable regionand CDR sequences may comprise one, two, three, four or more amino acidinsertions, deletions or substitutions. Where substitutions are made,preferred substitutions will be conservative modifications. Families ofamino acid residues having similar side chains have been defined in theart. These families include amino acids with basic side chains (e.g.,lysine, arginine, histidine), acidic side chains (e.g., aspartic acid,glutamic acid), uncharged polar side chains (e.g. glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolarside chains (e.g., alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine), beta-branched side chains (e.g. threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Thus, one or more amino acidresidues within the CDR regions of an antibody of the invention can bereplaced with other amino acid residues from the same side chain familyand the altered antibody can be tested for retained function (i.e., theproperties set forth herein) using the assays described herein.

The term “identity” or “identical”, when used in a relationship betweenthe sequences of two or more polypeptides, refers to the degree ofsequence relatedness between polypeptides, as determined by the numberof matches between strings of two or more amino acid residues.“Identity” measures the percent of identical matches between the smallerof two or more sequences with gap alignments (if any) addressed by aparticular mathematical model or computer program (i.e., “algorithms”).Identity of related polypeptides can be readily calculated by knownmethods. Such methods include, but are not limited to, those describedin Computational Molecular Biology, Lesk, A. M., ed., Oxford UniversityPress, New York, 1988; Biocomputing: Informatics and Genome Projects,Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis ofSequence Data, Part 1, Griffin, A. M., and Griffin, H. G., eds., HumanaPress, New Jersey, 1994; Sequence Analysis in Molecular Biology, vonHeinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M.and Devereux, J., eds., M. Stockton Press, New York, 1991; and Carilloet al., SIAM J. Applied Math. 48, 1073 (1988).

Preferred methods for determining identity are designed to give thelargest match between the sequences tested. Methods of determiningidentity are described in publicly available computer programs.Preferred computer program methods for determining identity between twosequences include the GCG program package, including GAP (Devereux etal., Nucl. Acid. Res. 12, 387 (1984); Genetics Computer Group,University of Wisconsin, Madison, Wis.), BLASTP, BLASTN, and FASTA(Altschul et al., J. Mol. Biol. 215, 403-410 (1990)). The BLASTX programis publicly available from the National Center for BiotechnologyInformation (NCBI) and other sources (BLAST Manual, Altschul et al.NCB/NLM/NIH Bethesda, Md. 20894; Altschul et al., supra). The well knownSmith Waterman algorithm may also be used to determine identity.

The sequences of the CDRs of the antibodies according to the invention,according to AbM (Oxford Molecular's AbM antibody modelling softwaredefinition), Kabat and Chothia definitions systems, have been summarizedin Table A below. The amino acids sequences described herein arenumbered according to Abm, Kabat and Chothia numbering systems. Whileany suitable numbering system may be used to designated CDR regions, inthe absence of any other indication, the numbering used herein is Abm.Such numbering has been established using the following indications:CDR-L1: Start: approx residue 24, residue before: always a Cys, residueafter: always a Trp (typically Trp-Tyr-Gln, but also, Trp-Leu-Gln,Trp-Phe-Gln, Trp-Tyr-Leu), length: 10 to 17 residues; CDR-L2: Start:always 16 residues after the end of L1, Residues before: generallyIle-Tyr (but also, Val-Tyr, Ile-Lys, Ile-Phe), Length: always 7residues; CDR-L3, Start: always 33 residues after end of L2, Residuebefore: always Cys, Residues after: always Phe-Gly-Xaa-Gly, Length: 7 to11 residues; CDR-H1, Start: approx residue 26 (always 4 after a Cys)(Chothia/AbM definition, the Kabat definition starts 5 residues later),Residues before: always Cys-Xaa-Xaa-Xaa, Residues after: always a Trp(typically Trp-Val, but also, Trp-Ile, Trp-Ala), Length: 10 to 12residues (AbM definition, Chothia definition excludes the last 4residues); CDR-H2, Start: always 15 residues after the end of Kabat/AbMdefinition of CDR-H1, Residues before: typically Leu-Glu-Trp-Ile-Gly(but a number of variations, Residues afterLys/Arg-Leu/Ile/Val/Phe/Thr/Ala-Thr/Ser/Ile/Ala), Length: Kabatdefinition 16 to 19 residues; AbM (and Chothia) definition ends 7residues earlier; CDR-H3, Start: always 33 residues after end of CDR-H2(always 2 after a Cys), Residues before: always Cys-Xaa-Xaa (typicallyCys-Ala-Arg), Residues after: always Trp-Gly-Xaa-Gly, Length: 3 to 25residues.

The sequences of the variable chains of the antibodies according to theinvention are listed in Table B below, signal peptide sequence isrepresented in italics, and the CDRs are provided in bold. The term x/xindicates that any of the two indicated amino acids can be present atthe particular amino acid residue, for instance, the term F/S means thatat the given position, the amino acid can be either phenylalanine orserine. In any embodiment herein, a VL or VH sequence can be specifiedor numbered so as to contain or lack the signal peptide or any partthereof.

In an embodiment, the antibodies of the invention are of the human ormouse IgG1 isotype. In another embodiment, the antibodies of theinvention are of the human IgG4 isotype In an embodiment, the antibodiesof the invention are antibody fragments that retain their binding and/orfunctional properties.

TABLE A HCDR1 HCDR2 HCDR3 CDR SEQ SEQ SEQ mAb definition ID Sequence IDSequence ID Sequence 23C8 Abm 28 GYSFTGYFMH 29 RINPYNGATS 30 DDGGNYPFDYChotia GYSFTG RINPYNGATS DDGGNYPFDY Kabat GYFMH RINPYNGATSY DDGGNYPFDYNQNFKD 29H3 Abm 12 GYSITSGYSWH 13 YIHYSGITN 14 DGYYGMDY Chotia GYSITSGYIHYSGITN DGYYGMDY Kabat SGYSWH YIHYSGITNYNP DGYYGMDY SLRS 28F11 Abm 20GYSFTGYYIH 21 RINPYYGAT 22 STKLGYLDY Chotia GYSFTG RINPYYGAT STKLGYLDYKabat GYYIH RINPYYGATSN STKLGYLDY QNFKD 31C3 Abm 4 GYSFTAYYMH 5RINPYNGATS 6 SGGNTYFDY Chotia GYSFTA RINPYNGATS SGGNTYFDY Kabat AYYMHRINPYNGATSY SGGNTYFDY NRNFKD 34A3 Abm 36 GYVFTTYSIY 37 YIDPYNGDTS 38EGNYYGYFDY Chotia GYVFTT YIDPYNGDTS EGNYYGYFDY Kabat TYSIY YIDPYNGDTSYEGNYYGYFDY NQKFKG

TABLE B SEQ Antibody ID portion NO 23C8 VL 27 MSVPTQVLGL LLLWLTGARCDIQMTQSPAS LSASVGETVT ITCRASENIY SYLAWYQQKQ GKSPQLLVYY AKTLAEGVPSRFSGSGTGTQ FSLKINSLQP EDFGSYYCQH HYGTPYTFGG GTKLEIK 23C8 VH 26MGWSWIFLFL LSGTAGVLSE VQLQQSGPEL VKPGASVKIS CKASGYSFTG YFMHWVKQSHVKSLEWIGRI NPYNGATSYN QNFKDKASLT VDKSSSTSYM ELHSLTSEDS AVYYCVRDDGGNYPFDYWGQ GTTLTVS 28F11 VL 19 MSVPTQVLGL LLLWLTGARC DIQMTQSPASLSASVGETVT ITCRASENIY SNLAWYQQKQ GKSPQLLIYN AKTLAEGVPS RFSGSGSGTQYFLKINSLQP EDFGSYYCQH HYGTPFTFGG GTKLEIK 28F11 VH 18 MGWSWIFLFLLSGTAGVLSE VQLQQSGPEL VKPGASVKIS CKASGYSFTG YYIHWVKQSH VKSLEWIGRINPYYGATSNQ NFKDKANLTV DKSSSTAYME LHSLTSDDSA VYYCARSTKL GYLDYWGQGT TLTVS29H3 VL 11 MDFQTQVFVF VLLWLSGVDG DIVMTQSQKF MSTSVGDRVS ITCKASQNVRTSVAWYQQKP GQSPKALIYL ASNRHTGVPD RFTGSGSGTD FTLTVSNIQS EDLADYFCLQHWNYPYTFGG GTKLEIK 29H3 VH 10 MRVLILLCLF TAFPGILSDV QLQESGPDLVKPSQSLSLTC TVTGYSITSG YSWHWIRQFL GNKLEWMGYI HYSGITNYNP SLRSRISFTRDTSKNQFFLQ LNSVTTEDTA TYYCARDGYY GMDYWGQGTS VTVS 31C3 VL 3 MSVPTQVLGLLLLWLTGARC DIQMTQSPAS LSASVGETVT ITCRASENIY SSLAWYQQKQ GKSPQLLVYNAKTLAEGVPS RFSGSGSGTQ F/SSLKINSLQP EDFGTYYCQH HYGTPPTFGG GTKLEIK 31C3 VH2 MGWSWIFLFL LSGTAGVLSE VQLQQSGPEL VKPGASVKIS CKPSGYSFTA YYMHWVKQSHVKSLEWIGRI NPYNGATSYN RNFKDKASLT VDKSSSTAYM ELHSLTSEDS AVYYCARSGGNTYFDYWGQG TTLTVS 34A3 VL 35 MDFQVQIFSF LLMSASVIMS RGQIVLTQSP ALMSASPGEKVTMTCSASSSV SYMFWYQQKP RSSPKPWIYL TSNLASGVPA RFSGSGSGTS YSLTISSMEAEDAATYYCQQ WTGNPPTFGG GTKLEIK 34A3 VH 34 MEWRWIFLFL LSGTTGVHSEIQLQQSGPEL VKPGASVKVS CKASGYVFTT YSIYWVKQSH GKSLEWIGYI DPYNGDTSYNQKFKGKATLT VDKSSSTAYM HLNSLTSEDS TVYYCAREGN YYGYFDYWGQ GTTLTVS

The sequencing of light and heavy chains of the anti-TLR3 antibodiesaccording to the invention led to the identification of the genesrearrangement involved in the generation of such antibodies, assummarized in Table C below. (The gene sequences indicated are can beretrieved at www.ncbi.nlm nih.gov/igblast/showGermline.cgi. FIGS. 18Aand 18B represent the phylogenetic trees (generated by Phylip'sDrawtree) of the light and heavy chains, indicating a high degree ofhomology between antibodies 28F11, 31C3 and 23C8.

TABLE C Light Chain Heavy Chain V gene J gene V gene J gene 29H3 VK19-14 JK2 VH3 VH36-60.a1.85 JH4 SEQ ID NO: 42 SEQ ID NO: 45 SEQ ID NO:46 SEQ ID NO: 50 34A3 VK aq4 (VK) JK2 VH1 VH J558.1 JH2 SEQ ID NO: 43SEQ ID NO: 45 SEQ ID NO: 47 SEQ ID NO: 49 23C8 VK 12-41 or 12-44 JK2 VH1VH J558.2 JH2 SEQ ID NO: 44 SEQ ID NO: 45 SEQ ID NO: 48 SEQ ID NO: 4931C3 VK 12-41 or 12-44 JK2 VH1 VH J558.2 JH2 SEQ ID NO: 44 SEQ ID NO: 45SEQ ID NO: 48 SEQ ID NO: 49 28F11 VK 12-41 or 12-44 JK2 VH1 VH J558.2JH2 SEQ ID NO: 44 SEQ ID NO: 45 SEQ ID NO: 48 SEQ ID NO: 49

Table D below provides the percentage sequence identity between thedifferent CDRs for each antibody, in amino acids.

TABLE D 31C3 29H3 23C8 28F11 34A3 CDR VL 31C3 100.00 33.33 88.89 92.5937.04 29H3 100.00 33.33 29.63 29.63 23C8 100.00 88.89 33.33 28F11 100.0033.33 34A3 100.00 CDR1 VL 31C3 100.00 45.45 90.91 90.91 27.27 29H3100.00 45.45 27.27 27.27 23C8 100.00 90.91 27.27 28F11 100.00 36.36 34A3100.00 CDR2 VL 31C3 100.00 14.29 85.71 100.00 28.57 29H3 100.00 14.2914.29 42.86 23C8 100.00 85.71 28.57 28F11 100.00 28.57 34A3 100.00 CDR3VL 31C3 100.00 33.33 88.89 85.71 33.33 29H3 100.00 44.44 27.27 22.2223C8 100.00 88.89 44.44 28F11 100.00 44.44 34A3 100.00 CDR VH 31C3100.00 39.47 78.38 70.27 50.00 29H3 100.00 44.74 39.47 39.47 23C8 100.0070.27 52.63 28F11 100.00 47.37 34A3 100.00 CDR1 VH 31C3 100.00 54.5580.00 80.00 45.45 29H3 100.00 63.64 63.64 45.45 23C8 100.00 80.00 45.4528F11 100.00 54.55 34A3 100.00 CDR2 VH 31C3 100.00 35.29 94.12 82.3564.71 29H3 100.00 35.29 29.41 35.29 23C8 100.00 88.24 70.59 28F11 100.0058.82 34A3 100.00 CDR3 VH 31C3 100.00 40.00 50.00 44.44 40.00 29H3100.00 40.00 30.00 40.00 23C8 100.00 30.00 30.00 28F11 100.00 20.00 34A3100.00

Table E provides the identity percentage has been calculated between thedifferent VL and VH (italics) nucleotide sequences for each antibodyusing LALIGN software.

TABLE E VH VL 23C8 28F11 29H3 31C3 34A3 23C8 100 93.5 60.6 94.9 87.328F11 97.6 100 60.4 94.4 87.8 29H3 65.8 65.2 100 61.0 60.6 31C3 96.195.5 63.9 100 89.1 34A3 64.6 65.3 67.2 64.0 100

Table F provides the identity percentage has been calculated between thedifferent VL and VH (italics) amino acid sequences using LALIGNsoftware.

TABLE F VH VL 23C8 28F11 29H3 31C3 34A3 23C8 100 89.8 48.9 92.7 79.128F11 94.5 100 47.0 91.2 79.6 29H3 54.3 54.3 100 47.8 48.1 31C3 96.195.3 55.1 100 79.6 34A3 52.7 53.5 55.4 54.3 100Fragments and Derivatives of the present Monoclonal Antibodies

Fragments and derivatives of antibodies of this invention (which areencompassed by the term “antibody” or “antibodies” as used in thisapplication, unless otherwise stated or clearly contradicted bycontext), preferably a 31C3, 29H3, 23C8, 28F11 or 34A3-like antibody,can be produced by techniques that are known in the art. “Fragments”comprise a portion of the intact antibody, generally the antigen bindingsite or variable region. Examples of antibody fragments include Fab,Fab′, Fab′-SH, F (ab′) 2, and Fv fragments; diabodies; any antibodyfragment that is a polypeptide having a primary structure consisting ofone uninterrupted sequence of contiguous amino acid residues (referredto herein as a “single-chain antibody fragment” or “single chainpolypeptide”), including without limitation (1) single-chain Fvmolecules (2) single chain polypeptides containing only one light chainvariable domain, or a fragment thereof that contains the three CDRs ofthe light chain variable domain, without an associated heavy chainmoiety and (3) single chain polypeptides containing only one heavy chainvariable region, or a fragment thereof containing the three CDRs of theheavy chain variable region, without an associated light chain moiety;and multispecific antibodies formed from antibody fragments.

Fragments of the present antibodies can be obtained using standardmethods. For instance, Fab or F (ab′) 2 fragments may be produced byprotease digestion of the isolated antibodies, according to conventionaltechniques. It will be appreciated that immunoreactive fragments can bemodified using known methods, for example to slow clearance in vivo andobtain a more desirable pharmacokinetic profile the fragment may bemodified with polyethylene glycol (PEG). Methods for coupling andsite-specifically conjugating PEG to a Fab′ fragment are described in,for example, Leong et al, 16 (3): 106-119 (2001) and Delgado et al, Br.J. Cancer 73 (2): 175-182 (1996), the disclosures of which areincorporated herein by reference.

Alternatively, the DNA of a hybridoma producing an antibody of theinvention, preferably a 31C3, 29H3, 23C8, 28F11 or 34A3-like antibody,may be modified so as to encode a fragment of the invention. Themodified DNA is then inserted into an expression vector and used totransform or transfect an appropriate cell, which then expresses thedesired fragment.

In certain embodiments, the DNA of a hybridoma producing an antibody ofthis invention, preferably a 31C3, 29H3, 23C8, 28F11 or 34A3-likeantibody, can be modified prior to insertion into an expression vector,for example, by substituting the coding sequence for human heavy- andlight-chain constant domains in place of the homologous non-humansequences (e.g., Morrison et al., PNAS pp. 6851 (1984)), or bycovalently joining to the immunoglobulin coding sequence all or part ofthe coding sequence for a non-immunoglobulin polypeptide. In thatmanner, “chimeric” or “hybrid” antibodies are prepared that have thebinding specificity of the original antibody. Typically, suchnon-immunoglobulin polypeptides are substituted for the constant domainsof an antibody of the invention.

Thus, according to another embodiment, the antibody of this invention,preferably a 31C3, 29H3, 23C8, 28F11 or 34A3-like antibody, is humanized“Humanized” forms of antibodies according to this invention are specificchimeric immunoglobulins, immunoglobulin chains or fragments thereof(such as Fv, Fab, Fab′, F (ab′) 2, or other antigen-binding subsequencesof antibodies) which contain minimal sequence derived from the murineimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from acomplementary-determining region (CDR) of the recipient are replaced byresidues from a CDR of the original antibody (donor antibody) whilemaintaining the desired specificity, affinity, and capacity of theoriginal antibody.

In some instances, Fv framework residues of the human immunoglobulin maybe replaced by corresponding non-human residues. Furthermore, humanizedantibodies can comprise residues that are not found in either therecipient antibody or in the imported CDR or framework sequences. Thesemodifications are made to further refine and optimize antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof the original antibody and all or substantially all of the FR regionsare those of a human immunoglobulin consensus sequence. The humanizedantibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details see Jones et al., Nature, 321, pp.522 (1986); Reichmann et al, Nature, 332, pp. 323 (1988); Presta, Curr.Op. Struct. Biol., 2, pp. 593 (1992); Verhoeyen et Science, 239, pp.1534; and U.S. Pat. No. 4,816,567, the entire disclosures of which areherein incorporated by reference.) Methods for humanizing the antibodiesof this invention are well known in the art.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is very important to reduceantigenicity. According to the so-called “best-fit” method, the sequenceof the variable domain of an antibody of this invention is screenedagainst the entire library of known human variable-domain sequences. Thehuman sequence which is closest to that of the mouse is then accepted asthe human framework (FR) for the humanized antibody (Sims et al., J.Immunol. 151, pp. 2296 (1993); Chothia and Lesk, J. Mol. 196, pp. 901).Another method uses a particular framework from the consensus sequenceof all human antibodies of a particular subgroup of light or heavychains. The same framework can be used for several different humanizedantibodies (Carter et al., PNAS 89, pp. 4285 (1992); Presta et J.Immunol., 51, p. 1993)).

It is further important that antibodies be humanized with retention ofhigh affinity for TLR3 receptors and other favorable biologicalproperties. To achieve this goal, according to a preferred method,humanized antibodies are prepared by a process of analysis of theparental sequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional structures ofselected candidate immunoglobulin sequences. Inspection of thesedisplays permits analysis of the likely role of the residues in thefunctioning of the candidate immunoglobulin sequence, i.e., the analysisof residues that influence the ability of the candidate immunoglobulinto bind its antigen. In this way, FR residues can be selected andcombined from the consensus and import sequences so that the desiredantibody characteristic, such as increased affinity for the targetantigen (s), is achieved. In general, the CDR residues are directly andmost substantially involved in influencing antigen binding.

Another method of making “humanized” monoclonal antibodies is to use aXenoMouse (Abgenix, Fremont, Calif.) as the mouse used for immunization.A XenoMouse is a murine host according to this invention that has hadits immunoglobulin genes replaced by functional human immunoglobulingenes. Thus, antibodies produced by this mouse or in hybridomas madefrom the B cells of this mouse, are already humanized. The XenoMouse isdescribed in U.S. Pat. No. 6,162,963, which is herein incorporated inits entirety by reference.

Human antibodies may also be produced according to various othertechniques, such as by using, for immunization, other transgenic animalsthat have been engineered to express a human antibody repertoire(Jakobovitz et Nature 362 (1993) 255), or by selection of antibodyrepertoires using phage display methods. Such techniques are known tothe skilled person and can be implemented starting from monoclonalantibodies as disclosed in the present application.

The antibodies of the present invention, preferably a 31C3, 29H3, 23C8,28F11 or 34A3-like antibody, may also be derivatized to “chimeric”antibodies (immunoglobulins) in which a portion of the heavy/lightchain(s) is identical with or homologous to corresponding sequences inthe original antibody, while the remainder of the chain (s) is identicalwith or homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired biological activity and binding specificity (Cabilly et al.,supra; Morrison et al., Proc. Natl. Acad. Sci. U.S.A, pp. 6851 (1984)).

While the present antibodies have the ability to bind TLR3 andoptionally further inhibit TLR3 signaling, they can also be used forother purposes such as conjugation to a toxin is useful for targetedkilling cells expressing TLR3 polypeptides, e.g. certain tumor cellssuch as melanomas or those of the breast, lung, esophagus, stomach,larynx, kidney, or cervix, or potentially virally infected cells orcells involved in inflammation. In such embodiments, typically a sampleis obtained (e.g. a biopsy for tumors) will be performed initially toassess whether the cells (e.g. tumor cells, infected cells, cellsinvolved in inflammation, etc.) express TLR3, e.g., using the detectionmethods described herein. If TLR3 is indeed detected on the surface ofthe tumor cells, then, cytotoxic antibodies can be administered. Thecytotoxic antibody is then internalized by the cell and the toxin isreleased inside of the cell, selectively killing that cell. Suchantibodies will therefore be used in methods of treating of cancers andtumors, include but not limited to biliary tract cancer; brain cancer;breast cancer; cervical cancer; choriocarcinoma; colon cancer;endometrial cancer; esophageal cancer; gastric (stomach) cancer;intraepithelial neoplasms; leukemias; lymphomas; liver cancer; lungcancer (e.g., small cell and non-small cell); melanoma; neuroblastomas;oral cancer; ovarian cancer; pancreatic cancer; prostate cancer; rectalcancer; renal (kidney) cancer; sarcomas; skin cancer; testicular cancer;thyroid cancer; as well as other carcinomas and sarcomas. Cancers can beprimary or metastatic.

One effective approach for enhancing the anti-tumor-potency ofantibodies involves linking cytotoxic drugs or toxins to mAbs that arecapable of being internalized by a target cell. These agents are termedantibody-drug conjugates (ADCs) and immunotoxins, respectively. Uponadministration to a patient, ADCs and immunotoxins bind to target cellsvia their antibody portions and become internalized, allowing the drugsor toxins to exert their effect. See, for example, U.S. Patent Appl.Publ. Nos. US2005/0180972 A1, US2005/0123536 A1. See also, for example,Hamblett et al., Clin Canc Res, 10:7063-7070, Oct. 15, 1999, Law et al.,Clin Canc Res, 10:7842-7851, Dec. 1, 2004, Francisco et al., Neoplasia,102(4):1458-1465, Aug. 15, 2003, Russell et al., Clin Canc Res,11:843-852, Jan. 15, 2005, Doronina et al., Nat Biotech, 21(7):778-784,July 2003, all of which are hereby incorporated by reference herein intheir entirety.

Cytotoxic antibodies can be prepared using described techniques (Carteret al, Cancer Journal, Volume 14, Number 3, May/June 2008, or Ravi etal, Accounts of Chemical Research 98-17 Jan. 2008 Vol. 41, No. 1).

Preferred toxic or cytotoxic peptides or small molecules include anycompound that can slow down, halt, or reverse the proliferation ofcells, decrease their activity in any detectable way, or directly orindirectly kill them. Preferably, toxic or cytotoxic compounds work bydirectly killing the cells, by provoking ADCC or otherwise. As usedherein, a toxic peptide can include any peptide, polypeptide, orderivative of such, including peptide- or polypeptide-derivatives withunnatural amino acids or modified linkages. A toxic small molecule caninclude any toxic compound or element, preferably with a size of lessthan 10 kD, 5 kD, 1 kD, 750 D, 600 D, 500 D, 400 D, 300 D, or smaller.

Conjugation to a detectable moiety is useful, inter alia, when anantibody of the invention is used for diagnostic purposes. Such purposesinclude, but are not limited to, assaying biological samples, e.g., ablood sample or tissue biopsy, for the presence of TLR3-expressingcells, and detecting the presence, level, or activity of TLR3-expressingcells in an individual. Such assay and detection methods can be used inthe diagnostic/therapeutic methods of the invention, e.g., involvingdetecting TLR3 expression in cells of a patient and if the patient'scells are determined to express TLR3, subsequently administering a TLR3modulating antibody of the invention.

In certain embodiments, the present antibodies are used to purifyTLR3-expressing cells from a biological sample. Biological samples canbe obtained from a patient, e.g. for diagnostic or ex vivo therapeuticpurposes, or from individuals or non-human primates to obtain a sourceof such cells for research purposes.

In one such embodiment, labeled antibodies of the invention can be usedin FACS sorting to purify or isolate TLR3-expressing cells from abiological sample. Alternatively, in some embodiments conjugation of anantibody of this invention to a solid support can be useful as a toolfor affinity purification of cells bearing a TLR3 receptor on their cellsurface from a biological sample, such as a blood sample or cells from atissue biopsy from an individual. This method of purification is anotheralternate embodiment of the present invention, as is the resultingpurified population of cells.

Regardless of the method used to isolate or purify the TLR3-expressingcells, the ability to do so is useful for numerous purposes, e.g. todiagnose a TLR3-associated disorder by assessing the number or activityof TLR3-expressing cells, e.g., prior to administration of anti-TLR3antibodies as described herein. Further, purified TLR3-expressing cellsare useful in a research context, e.g., to better characterize the cellsand their various properties and behaviors, as well as to identifycompounds or methods that can be used to modulate their behavior,activity, survival, or proliferation.

Compositions and Uses in Therapy, Diagnostics and Prognostics

As demonstrated herein, the antibodies of the invention are particularlyeffective at modulating the activity of TLR3-expressing cells comprisingthe polypeptides and, consequently, the activity or behavior of thecells expressing the polypeptides, e.g., TLR3-expressing immune cells,DC, myeloid DC, etc. In certain embodiments, the antibodies inhibitTLR3, e.g., by blocking TLR3 signaling, optionally without blocking theinteraction of an antigen or ligand such as dsRNA to the receptor,thereby inhibiting the proliferation or activation of the cells. Thecomposition further comprises a pharmaceutically acceptable carrier.Such compositions are also referred to as “antibody compositions” of theinvention. In one embodiment, antibody compositions of this inventioncomprise an antibody disclosed in the antibody embodiments above. Theantibody 31C3, 29H3, 23C8, 28F11 or 34A3 is included within the scope ofantibodies that may be present in the antibody compositions of thisinvention.

The invention further provides a method of modulating TLR3-expressingcell activity in a patient in need thereof, comprising the step ofadministering to said patient a composition according to the invention.In one embodiment, the TLR3-expressing cell activity is inhibited,wherein the patient has a disease or disorder wherein such inhibitionmay promote, enhance, and/or induce a therapeutic effect (or promotes,enhances, and/or induces such an effect in at least a substantialproportion of patients with the disease or disorder and substantiallysimilar characteristics as the patient, as may determined by, e. g.,clinical trials).

In other embodiments, the method may comprise the additional step ofadministering to said patient an appropriate additional therapeuticagent useful in treatment or prevention of the disease from which thepatient suffers or is susceptible to; examples of such agents include animmunomodulatory agent, a hormonal agent, an anti-inflammation drug, asteroid, an immune system suppressor, a corticosteroid, an antibiotic,an anti-viral or an adjunct compound. Such additional agents can beadministered to a patient as a single dosage form together with saidantibody, or as a separate dosage form. The dosage of the antibody (orantibody and the dosage of the additional therapeutic agentcollectively) are sufficient to detectably induce, promote, and/orenhance a therapeutic response in the patient. Where administeredseparately, the antibody, fragment, or derivative and the additionaltherapeutic agent are desirably administered under conditions (e.g.,with respect to timing, number of doses, etc.) that result in adetectable combined therapeutic benefit to the patient.

Pharmaceutically acceptable carriers that may be used in thesecompositions include, but are not limited to, ion exchangers, alumina,aluminum stearate, lecithin, serum proteins, such as human serumalbumin, buffer substances such as phosphates, glycine, sorbic acid,potassium sorbate, partial glyceride mixtures of saturated vegetablefatty acids, water, salts or electrolytes, such as protamine sulfate,disodium hydrogen phosphate, potassium hydrogen phosphate, sodiumchloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodiumcarboxymethylcellulose, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, polyethylene glycol andwool fat. The antibodies of this invention may be employed in a methodof modulating, e.g inhibiting, the activity of TLR3-expressing cells ina patient. This method comprises the step of contacting said compositionwith said patient. Such method will be useful for both prophylaxis andtherapeutic purposes.

For use in administration to a patient, the composition will beformulated for administration to the patient. The compositions of thepresent invention may be administered orally, parenterally, byinhalation spray, topically, rectally, nasally, buccally, vaginally orvia an implanted reservoir. The used herein includes subcutaneous,intravenous, intramuscular, intra-articular, intra-synovial,intrasternal, intrathecal, intrahepatic, intralesional and intracranialinjection or infusion techniques.

Sterile injectable forms of the compositions of this invention may beaqueous or an oleaginous suspension. These suspensions may be formulatedaccording to techniques known in the art using suitable dispersing orwetting agents and suspending agents. The sterile injectable preparationmay also be a sterile injectable solution or suspension in a non-toxicparenterally acceptable diluent or solvent, for example as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that may beemployed are water, Ringer's solution and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium. For this purpose, any bland fixed oilmay be employed including synthetic mono-or diglycerides. Fatty acids,such as oleic acid and its glyceride derivatives are useful in thepreparation of injectables, as are natural pharmaceutically-acceptableoils, such as olive oil or castor oil, especially in theirpolyoxyethylated versions. These oil solutions or suspensions may alsocontain a long-chain alcohol diluent or dispersant, such ascarboxymethyl cellulose or similar dispersing agents that are commonlyused in the formulation of pharmaceutically acceptable dosage formsincluding emulsions and suspensions. Other commonly used surfactants,such as Tweens, Spans and other emulsifying agents or bioavailabilityenhancers which are commonly used in the manufacture of pharmaceuticallyacceptable solid, liquid, or other dosage forms may also be used for thepurposes of formulation.

The compositions of this invention may be orally administered in anyorally acceptable dosage form including, but not limited to, capsules,tablets, aqueous suspensions or solutions. In the case of tablets fororal use, carriers commonly used include lactose and corn starch.Lubricating agents, such as magnesium stearate, are also typicallyadded. For oral administration in a capsule form, useful diluentsinclude, e.g., lactose. When aqueous suspensions are required for oraluse, the active ingredient is combined with emulsifying and suspendingagents. If desired, certain sweetening, flavoring or coloring agents mayalso be added.

Alternatively, the compositions of this invention may be administered inthe form of suppositories for rectal administration. These can beprepared by mixing the agent with a suitable non-irritating excipientthat is solid at room temperature but liquid at rectal temperature andtherefore will melt in the rectum to release the drug. Such materialsinclude cocoa butter, beeswax and polyethylene glycols.

The compositions of this invention may also be administered topically,especially when the target of treatment includes areas or organs readilyaccessible by topical application, including diseases of the eye, theskin, or the lower intestinal tract. Suitable topical formulations arereadily prepared for each of these areas or organs. For topicalapplications, the compositions may be formulated in a suitable ointmentcontaining the active component suspended or dissolved in one or morecarriers. Carriers for topical administration of the compounds of thisinvention include, but are not limited to, mineral oil, liquidpetrolatum, white petrolatum, propylene glycol, polyoxyethylene,polyoxypropylene compound, emulsifying wax and water. Alternatively, thecompositions can be formulated in a suitable lotion or cream containingthe active components suspended or dissolved in one or morepharmaceutically acceptable carriers. Suitable carriers include, but arenot limited to, mineral oil, sorbitan monostearate, polysorbate 60,cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol andwater.

Topical application for the lower intestinal tract can be effected in arectal suppository formulation (see above) or in a suitable enemaformulation. Patches may also be used.

The compositions of this invention may also be administered by nasalaerosol or inhalation. Such compositions are prepared according totechniques well-known in the art of pharmaceutical formulation and maybe prepared as solutions in saline, employing benzyl alcohol or othersuitable preservatives, absorption promoters to enhance bioavailability,fluorocarbons, and/or other conventional solubilizing or dispersingagents.

Several monoclonal antibodies have been shown to be efficient inclinical situations, such as Rituxan Herceptin (Trastuzumab) or Xolair(Omalizumab), and similar administration regimens (i.e., formulationsand/or doses and/or administration protocols) may be used with theantibodies of this invention. Schedules and dosages for administrationof the antibody in the pharmaceutical compositions of the presentinvention can be determined in accordance with known methods for theseproducts, for example using the manufacturers' instructions. Forexample, an antibody present in a pharmaceutical composition of thisinvention can be supplied at a concentration of 10 mg/mL in either 100mg (10 mL) or 500 mg (50 mL) single-use vials. The product is formulatedfor IV administration in 9.0 mg/mL sodium chloride, 7.35 mg/mL sodiumcitrate dihydrate, 0.7 mg/mL polysorbate 80, and Sterile Water forInjection. The pH is adjusted to 6.5. An exemplary suitable dosage rangefor an antibody in a pharmaceutical composition of this invention maybetween about 1 mg/m² and 500 mg/m². However, it will be appreciatedthat these schedules are exemplary and that an optimal schedule andregimen can be adapted taking into account the affinity and tolerabilityof the particular antibody in the pharmaceutical composition that mustbe determined in clinical trials.

According to another embodiment, the antibody compositions of thisinvention may further comprise another therapeutic agent, includingagents normally utilized for the particular therapeutic purpose forwhich the antibody is being administered. The additional therapeuticagent will normally be present in the composition in amounts typicallyused for that agent in a monotherapy for the particular disease orcondition being treated. Such therapeutic agents include, but are notlimited to anti-inflammation agents, steroids, immune systemsuppressors, antibiotics, antivirals and other antibodies and fragmentsthereof.

In another embodiment, two or more antibodies of this invention havingdifferent cross-reactivities, e.g. antibodies that specifically bind todistinct epitopes within the TLR3 polypeptide, are combined in a singlecomposition so as to target as many distinct TLR3 gene products aspossible, e.g. to account for diversity in the polypeptides within anindividual or in different patients, and to do so as efficaciously aspossible. In addition, an antibody composition of this invention maycomprise multiple antibodies that recognize a single TLR3 epitope. Suchcombinations would again provide wider utility in a therapeutic setting.

The invention also provides a method of modulating TLR3-expressing cellactivity in a patient in need thereof, comprising the step ofadministering a composition according to this invention to said patient.The method is more specifically directed at decreasing TLR3 cellactivity in patients having a disease in which decreased TLR3 cellactivity is beneficial (e.g., autoimmune diseases, inflammatorydiseases, infectious disease, viral infection), or which is caused orcharacterized by excessive TLR3 cell activity.

Diseases and conditions in which the present methods can be used includeall diseases where modulating TLR3 can be beneficial, including forexample diseases mediated or exacerbated partially or totally by TLR3signaling or by cytokines produced upon said TLR3 signaling. Inparticular, where antibodies that inhibit TLR3 signaling are used, suchdisorders include any disorders mediated or exacerbated partially ortotally by TLR3 signaling or by cytokines produced upon said TLR3signaling, including inter alia immune disorders such as inflammatorydiseases and autoimmune diseases. More specifically, the methods of thepresent invention are utilized for the treatment of a variety of immunedisorders and other diseases including, but not limited to autoimmunity,inflammation, allergy, asthma, infections (e.g. chronic infection, viralinfection) and sepsis. Examples of diseases which can be treated withthe antibodies that inhibit TLR3 signaling include, but are not limitedto arthritis, systemic lupus erythematosus, sepsis, asthma,osteoporosis, autoimmunity to central nervous system antigens,autoimmune diabetes, inflammatory bowel disease, autoimmune carditis,autoimmune hepatitis.

Other immune disorders treatable using the antibodies that inhibit TLR3signaling according to the invention include, inter alia, autoimmunedisorders and inflammatory disorders, including, but not limited to,Crohn's disease, Celiac disease, ulcerative colitis, irritable bowelsyndrome, acute disseminated encephalomyelitis (ADEM), Addison'sdisease, antiphospholipid antibody syndrome (APS), aplastic anemia,autoimmune hepatitis, Diabetes mellitus, Goodpasture's syndrome, Graves'disease, Guillain-Barre syndrome (GBS), Hashimoto's disease, lupuserythematosus, demyelinating conditions, Multiple sclerosis, Myastheniagravis, opsoclonus myoclonus syndrome (OMS), optic neuritis, Ord'sthyroiditis, pemphigus, cirrhosis, psoriasis, rheumatoid arthritis,Reiter's syndrome, Takayasu's arteritis, temporal arteritis, warmautoimmune hemolytic anemia, Wegener's granulomatosis, appendicitis,arteritis, arthritis, blepharitis, bronchiolitis, bronchitis, bursitis,cervicitis, cholangitis, cholecystitis, chorioamnionitis, colitis,conjunctivitis, cystitis, dacryoadenitis, dermatitis, dermatomyositis,encephalitis, endocarditis, endometritis, enteritis, enterocolitis,epicondylitis, epididymitis, fasciitis, fibrositis, gastritis,gastroenteritis, gingivitis, hepatitis, hidradenitis suppurativa,ileitis, iritis, laryngitis, mastitis, meningitis, myelitis,myocarditis, myositis, nephritis, omphalitis, oophoritis, orchitis,osteitis, otitis, pancreatitis, parotitis, pericarditis, peritonitis,pharyngitis, pleuritis, phlebitis, pneumonitis, proctitis, prostatitis,pyelonephritis, rhinitis, salpingitis, sinusitis, stomatitis, synovitis,tendonitis, tonsillitis, uveitis, vaginitis, vasculitis, and vulvitis.

The present antibodies can be included in kits. The kits may optionallyfurther contain any number of antibodies and/or other compounds, e.g.,1, 2, 3, 4, or any other number of therapeutic antibodies and/orcompounds. It will be appreciated that this description of the contentsof the kits is not limiting in any way. For example, the kit may containother types of therapeutic compounds. Preferably, the kits also includeinstructions for using the antibodies, e.g., detailing theherein-described methods.

Further aspects and advantages of this invention will be disclosed inthe following experimental section, which should be regarded asillustrative and not limiting the scope of this application.

EXAMPLES Materials and Methods

Interferon-alpha (IntronA™) was purchased from Schering Plough Corp.Tumor Cell lines: A375 malignant melanoma tumor cell lines (CRL-1619)are purchased from ATCC. Antibodies (antigen, supplier, reference):Anti-TLR3 antibody pAb, R&D Systems, ref. AF1487. Instrumentation:FACSCalibur™ flow cytometer (BD Biosciences).

Inhibition with Lentivirus shRNA

A lentivirus construction was made and produced by Vectalys (Toulouse,France), encoding short hairpin RNA (shRNA) targeting control humanTLR3. Tumor cells were infected with lentivirus preparation and furtherselected with puromycin to get stable shTLR3 A375 tumor cells.

Surface Plasmon Resonance (SPR)

(a) General Biacore T100 methods. SPR measurements were performed on aBiacore T100 apparatus (Biacore GE Healthcare) at 25° C. In all Biacoreexperiments HBS-EP+ buffer (Biacore GE Healthcare) or 10 mM sodiumacetate pH 5,6, 150 mM NaCl, 0.05% P20 served as running buffer andsensorgrams were analyzed with Biaevaluation 4.1 and Biacore T100Evaluation software. Recombinant human TLR3 was purchased from R&DSystems.

(b) Protein immobilization. Recombinant TLR3 protein was immobilizedcovalently to carboxyl groups in the dextran layer of a Biacore Series 5Sensor Chip CM5 (chip). The chip surface was activated with EDC/NHS(0.2M N-ethyl-N′-(3-dimethylaminopropyl) carbodiimidehydrochloride,0.05M N-hydroxysuccinimide (Biacore GE Healthcare)). Proteins werediluted to 10 μg/ml in coupling buffer (10 mM sodium acetate, pH 5.6)and injected until the appropriate immobilization level was reached(i.e. approximately 2000 RU for binding experiments and 600 RU foraffinity experiments). Deactivation of the remaining activated groupswas performed using 100 mM ethanolamine pH 8 (Biacore GE Healthcare).

(c) Antibody binding analysis was run using HBS-EP+ (neutral pH).Antibodies at a concentration of 10 μg/ml were injected for 2 min at aconstant flow rate of 10 μl/min over the immobilized proteins andallowed to dissociate for 3 min before regeneration by a ten secondinjection of 10 mM NaOH, 500 mM NaCl regeneration buffer. Blankcorrection was performed on line by co-injecting the soluble antibodiesonto the reference dextran flow cell.

(d) Competition assay in acidic buffer (pH 5,6). Flow rate was set to 10μl/min, the first antibody at a concentration of 50 μg/ml (or polyAU at100 μg/ml) was injected for 2 min, 3 times successively in order tosaturate the rhTLR3 surface. The second antibody was then injected for 2min also at 50 μg/ml and allowed to dissociate for 3 min beforeregeneration by a 15 second injection of 10 mM NaOH, 500 mM NaClregeneration buffer. Blank correction was also performed on line and thecurve using the saturating antibody (or nucleic acid) followed by aninjection of buffer subtracted to remove the signal due to thedissociation of the first complex. The resulting signal was compared tothat obtained by the injection of the second antibody directly onto therhTLR3 surface.

Luciferase Reporter Assay.

A reporter gene assay using as promoter ISRE (IFN-stimulated responseelement) and as reporter gene and protein luciferase was set up. A 293Tcell line (ATCC, #CRL-1573) was stably transfected with pISRE-lucplasmid (#219089—Stratagene), further selected as inducing optimalresponse to IFN-alpha stimulation and referred to as control 293T-ISRE.This cell line was further stably transfected with different plasmidspUNO-hTLR3 plasmid (#puno-htlr3—InVivogen) and referred to as293T-TLR3-ISRE. On day 0, cells are seeded at 4×10⁵ cells/mL in completeculture medium in 96-well culture plate (100 W/well). Cells are firstincubated at 37° C. for 20 hours, then 50 μL of medium are discarded andcells are activated with 100 μL/well final of increasing amounts ofpolyAU together with various concentrations of anti-TLR3 antibodies.Cells incubated with fresh medium will be used as background luciferaseactivity. Cells are incubated at 37° C. for 6 hours. 100 μL of freshlythawed Steady Glo (Promega) are added to each well, plates wereincubated 10 min at RT in the dark and the light emitted in each well isquantified as Count Per Second (CPS) on a gamma-counter (TopCount)apparatus.

Example 1 Generation of TLR3-Specific Monoclonal Antibodies Immunization#1

Primary Screen.

To obtain anti-TLR3antibodies, Balb/c mice were immunized with arecombinant human His-tagged TLR3 extracellular domain recombinantprotein (R&D systems, #1487-TR-050). Mice received oneprimo-immunisation with an emulsion of 50 μg TLR3 protein and CompleteFreund Adjuvant, intraperitoneally, a 2nd immunization with an emulsionof 50 μg TLR3 protein and Incomplete Freund Adjuvant, intraperitoneally,and three boosts with 10 μg TLR3 protein, intravenously. Immune spleencells were fused with X63.Ag8.653 immortalized B cells, and cultured inthe presence of irradiated spleen cells. 40 culture plates were obtainedand evaluated in a first screen for TLR3 binding using an ELISAdeveloped for detection of binding to TLR3. Briefly, His-taggedrecombinant TLR3 protein (R&D systems, #1487-TR-050) was coated onNi-NTA 96-wells plates (Qiagen). Supernatant (SN) from hybridoma cultureplates and incubated in TLR3-plates, and the presence of TLR3 binding Igwas revealed with goat anti-mouse F(ab) IgG-HRP. Positive supernatantswere selected and tested for lack of binding to TLR4. Briefly,His-tagged rec TLR4 protein (R&D systems, #3146-TR-050) were coated onNi-NTA 96-wells plates (Qiagen). SN from hybridoma culture plates wereincubated in TLR4-plates, and the presence of TLR4 binding Ig wasrevealed with goat anti-mouse F(ab) IgG-HRP. TLR4 was chosen as a 2^(nd)screen in order to discriminate among wells selected in the 1^(st)screen, where anti-His specific antibody from TLR3 specific antibodywere used. Secondly, given the homology between TLR3 and other membersof TLR family, the initial assessment in demonstrated that at least onecommercially available monoclonal antibody (mAb) indicated on itspackaging as specific for TLR3 protein nevertheless recognizedparaffin-embedded 293T cells stably transfected with TLR4.

Secondary Screen; Selection of Hybridomas of Interest.

168 supernatants were retained and tested in a further screen in aBiacore assay using rec TLR3 chips, followed by various assays formatsbased on binding to human TLR3-expressing 293T cells. A 293T cell line(ATCC, #CRL-1573), stably transfected with pISRE-luc plasmid(#219089—Stratagene), was further selected as inducing optimal responseto IFN-alpha stimulation and referred to as control 293T cells. Thiscell line was further stably transfected with pUNO-hTLR3 plasmid(#puno-htlr3—InVivogen), or pUNO-hTLR4 plasmid (#puno-tlr4—InVivogen)and referred to respectively as 293T-TLR3 and 293T-TLR4. Supernatantswere screened in a FACS based screen for binding to 293T-TLR3 cells withno binding to 293T control cells, and in parallel in an IHC screen forbinding to 293T-TLR3 cells as a frozen cell pellet, with no binding to293T-TLR4 cells. Wells selected in the IHC screen for binding to293T-TLR3 cells as a frozen cell pellet were also further tested in anIHC screen for binding to 293T-TLR3 cells as a paraffin embedded cellpellet. Briefly, for FACS screening, the presence of reacting antibodiesin supernanants were revealed by Goat anti-mouse polyclonal antibody(pAb) labeled with biotin, streptavidin labeled with PE. For IHCscreening, presence of reacting antibodies (Abs) in supernanants wererevealed by donkey anti-mouse pAb labeled with biotin (#715-065-150,Jackson Immunoresearch Laboratories), streptavidin labeled withperoxydase (#E2886, SIGMA) and revealed with DAB (#SK-4105, VectorLaboratories).

Cloning of Hybridomas of Potential Interest.

42 potentially interesting hybridomas selected from the initialscreening were cloned by limiting dilution techniques in 96-wellsplates, and 304 subclones were tested in a series of screens as follows.The 304 subclones were first evaluated in a screen for TLR3 bindingusing the same ELISA developed for detection of binding to TLR3, andpositive supernatants were selected and tested for lack of binding toTLR4 in ELISA assay, yielding 228 clones which were selective for TLR3.All supernatants yielding a ratio above 10 for DO obtained in TLR3 ELISAto DO obtained in TLR4 ELISA were selected as specific for TLR3. Amongthem were supernatants from well C3 of plate 31 (31C3), well H3 of plate29 (29H3), well E7 of plate 23 (23E7), well C8 of plate 23 (23C8) andwell F11 of plate 28 (28F11). 31C3, 29H3, 23C8 and 28F11 are of IgG1isotype.

Among the 304 clones, 63 clones, selected as issuing from preclonestested positive in frozen IHC, were also tested in a frozen IHC screenfor binding to 293T-TLR3 cells as a frozen cell pellet, with no bindingto 293T-TLR4 cells, yielding 31 positive clones in frozen IHC.

Among 71 clones positive in FACS staining and the 31 clones positives infrozen IHC, 41 clones were selected for cryopreservation from the 304initial clones.

Immunization #2

Primary Screen.

A further series of immunization were carried out in order to generatedifferent antibodies. Using an experimental setup similar to that of thefirst immunization series, Balb/c mice were immunized, immune spleencells were fused and cultured in the presence of irradiated spleencells. Culture plates were obtained and evaluated in a first screen forTLR3 binding using an ELISA developed for detection of binding to TLR3.263 clones out of 2840 were selected for the secondary screen.

Secondary Screen; Selection of Hybridomas of Interest.

263 supernatants were retained and tested in a further screen in aninhibition test on 293T-TLR3 cells. Clones having an inhibitory effectsuperior to 95% were selected. Among them were supernatants from well A3of plate 34 (34A3).

Example 2 TLR3 Modulation

Myeloid DC (MdDC) were obtained from PBMC by isolating PBMC from normalhealthy human donors. Monocytes were purified from PBMC using positiveselection with human CD14 microbeads (Miltenyi Biotech) followinggeneral instructions. Monocytes were further derived into DC (MdDC) by5-6 days incubation in human GM-CSF (Leucomax, SP) and human IL-4 (R&DSystems) at respectively 200 ng/ml and 20 ng/ml.

The resulting MdDC were then seeded at 10⁶ cells/ml, in duplicate, inflat bottom 96-well plates. Cells were activated for 20 hours in a finalvolume of 200 together with the anti-TLR3 antibodies at the indicatedconcentrations. Increasing amounts of polyAU (30, 100, 300 and 900μg/ml) were added to the wells to obtain a dose effect read-out.

Supernatants were collected after 20 h of stimulation, frozen at −20°C., and further assayed for IL-6 and IP-10 using Enzyme-linkedimmunosorbent assay. Cells were then harvested, stained for activationmarkers CD86 (with a.huCD86 mouse, IgG1, FITC, BD Biosciences, ref555657), with detection using FACSCanto™ flow cytometer (BDBiosciences).

FIGS. 1A-1C and 2A-2B illustrate the inhibitory properties of antibodies31C3 and 29H3 (both are of IgG1 isotype) in terms of CD86 cellularactivation and IL-6, IP-10 secretion. The anti-TLR3 antibodies 31C3.1and 29H3.7 antagonize, in vitro, TLR3-mediated myeloid DC activation,moreover, these antibodies effectively downregulated TLR3-mediatedCD83/CD86 expression and abrogated TLR3-mediated cytokine/chemokinesecretion, in particular IP-10 and IL-6.

F(ab)′2 fragments were generated from antibodies 31C3.1 and 29H3.7 bypapain cleavage and purification by ion-exchange chromatography on MonoQ5/50 GL, analyzed by SDS-PAGE, and tested for inhibition in terms ofCD86 cellular activation and cytokine secretion. F(ab)′2 fragments ofboth antibodies 31C3.1 and 29H3.7 effectively downregulatedTLR3-mediated CD83/CD86 expression and abrogated TLR3-mediatedcytokine/chemokine secretion, to a similar extent as full lengthantibodies. FIGS. 3A-3B show the results for antibody 31C3, whereF(ab)′2 fragments of antibody 31C3 and whole 31C3 IgG abrogateTLR3-mediated CD86 expression and IP-10 secretion to a similar extend;FIGS. 4A-4B show the same for F(ab)′2 fragments of antibody 29H3.

In a similar experiment, the ability of 28F11 and 23C8 antibodies toinhibit TLR3 signaling were assessed, compared to antibody 31C3. Theantibodies were tested at a concentration of 50 μg/ml in the presence ofan increasing dose of polyAU. The results are represented in FIGS. 14Aand 14B, underlining the inhibitory properties of the antibodiesaccording to the invention.

Antibody 34A3 has also been tested in a similar setting. This time, thecells (MdDC) are stimulated with a fixed dose of polyAU (300 μg/ml) andincreasing doses of the antibody is added to the medium. FIGS. 19A and19B illustrate the results for antibody 34A3, in comparison with 31C3 interms of IL-6 and IP-10 secretion.

Example 3 Bivalent Affinity

Binding properties of the antibodies 29H3.7, 23E7.3, 31C3.1 andcommercially available antibodies TLR3.7 (eBiosciences) and 40C1285(Abcam) were compared using the methods described for SPR, item c). FIG.5 shows that the binding affinity for TLR3 is significantly better inthe case of 29H3.7 and 31C3.1 than in the case of commercially availableantibodies.

Binding to TLR3 was determined at neutral (pH 7.2) and acid (pH 5.6)conditions, and K(D) values were calculated. The results (mean of 2 or 3experiments) are shown in Table 1. At neutral pH, 23E7, 29H3.7 and31C3.1 all showed strong and similar bivalent affinity (K_(D)) forrecombinant human TLR3 better than 100 picomolar (around 50 picomolar).Antibody TLR3.7 (eBiosciences) in comparison showed a significantlylower binding affinity. At acid pH, however, 23E7 lost considerablebinding affinity and its K_(D) was at about 4 nanomolar. The affinity of31C3, 23C8, 28F11 and 34A3 were measured in the same conditions in aseparate assay, the results are represented in Table 1 and in FIG. 15.These results indicate that the antibodies according to the inventionhave a high affinity, especially at acidic pH.

TABLE 1 Mean K_(D) (M) at Mean K_(D) (M) at Antibody pH 7.2 pH 5.623E7.3 3.51 * 10⁻¹¹ 4.32 * 10⁻⁹ 29H3.7 4.74 * 10⁻¹¹ 1.10 * 10⁻¹¹ 31C3.15.05 * 10⁻¹¹ 6.14 * 10⁻¹¹ TLR3.7 4.5* 10⁻⁸ 9* 10⁻⁹ 23C8 1.38* 10⁻¹⁰1.03* 10⁻¹¹ 28F11 6.50* 10⁻¹⁰ 3.35* 10⁻¹⁰ 34A3 3.01* 10⁻¹² 5.17* 10⁻¹¹

Example 4 Epitope Mapping

Epitope mapping at acid pH. Competition assays were conducted accordingto the methods described for SPR, item d) at pH 5.6. FIG. 6 illustratesthat antibodies 29H3.7 and 31C3.1 were able to bind TLR3 withoutcompeting with polyAU, similarly, FIGS. 16A and 16B illustrate thatantibodies 28F11, 34A3 and 23C8 were able to bind TLR3 without competingwith polyAU. 29H3.7, 31C3.1, 28F11, 34A3 and 23C8 are thus able toinhibit dsRNA signaling without competing for the epitope of dsRNA onTLR3. FIG. 7 illustrates the competition between antibodies 29H3.7 and31C3.1. As the binding to TLR3 by one antibody impairs the binding toTLR3 of the other (about 90% inhibition by one antibody of the other),one can conclude that these two antibodies compete for a similar epitopeon TLR3. Similarly, FIGS. 15A and 15B illustrate the competition betweenantibody 31C3 with antibodies TLR307, 23C3, 28F11 and 29H3. FIG. 15Aillustrates binding in the absence of any competing antibody whereasFIG. 15B illustrates the binding level after saturation of the hTLR3chip with 31C3. The loss in binding level reflects the hindrance causedby the 31C3 antibody, and underlines competition for binding between thetested antibodies. TLR3.7 binding level being not affected by 31C3competition underlines that the antibodies compete for differentepitopes, whereas the decrease in binding level for all of the otherantibodies underline that they all compete with 31C3 for a similarepitope.

Epitope mapping at neutral pH. Competition assays were conductedaccording to the methods described for SPR, at pH 7.2. Antibodies 29H3.7and 31C3.1 competed with one another for binding to TLR3 since bindingby one antibody impaired the binding to TLR3 of the other. Epitopemapping was explored also with commercial antibody TLR3.7 and antibody23E7.3. Neither of these antibodies competed for binding to TLR3 witheither of antibodies 29H3.7 and 31C3. However, antibody TLR3.7 didcompete for binding to TLR3 with antibody 23E7.3, indicating that theyhave overlapping binding sites.

The 34A3 antibody binding affinity was measured either alone on a rhTLR3chip or in conditions where the rhTLR3 chip was saturated with the 31C3antibody prior to 34A3 addition. The results are shown in FIG. 17, andprovide evidence that the two antibodies compete for binding to hTLR3,thus sharing a common epitopic determinant in view of their overlappingbinding sites.

FIG. 8 shows molecular surface maps of the extracellular domains of thehuman TLR3 protein, generated by computer modeling using SwissPdb Viewer4.0 (Guex and Peitsch (2007) Electrophoresis 18: 2714-2723) based ondata publicly available as data file 1ZIW from the Resource for StudyingBiological Macromolecules database from the Protein Data Bank (PDB)project of the European Bioinformatics Institute (Hinxton U.K.). TheC-terminal is on the left and the N-terminal on the right in this view,showing the face of TLR3 which is substantially glycosylation free, theother face being extensively masked by carbohydrates (see e.g. Choe etal. (2005) Science 309:581-585). The surface map on the right hand sidein FIG. 8 shows the electrostatic potential of the human TLR3polypeptide at neutral pH, mapped to the molecular surface. The mainareas of positive electrostatic potential are indicated with arrows,forming a general region of positive and neutral potential on theglycosylation free face of the C-terminal side. The glycosylation-freeface is believed to be accessible for interaction with ligands accessorymolecules or TLR3 monomers or other oligomer assemblies, and the regionof positive electrostatic potential on the C-terminal side (in thedarkest shading, the main positive areas indicated by arrows) is thoughtto correspond to the dsRNA binding region. These regions are also shownin FIG. 5 of Choe et al. (2005). The map on the left hand side indicatesamino acid residues determined by mutation studies to be involved indsRNA binding, where residues dsRNA binding was inhibited (>80%) bymutation of residues H539, N541, N466, R489, N517 or N540, and wheredsRNA binding was inhibited (>80%) by mutation of residues K117, K137and K139. The model proposed in Choe et al. (2005) is that TLR3 forms ahomodimer where the dimerization interface is proximal to the C-terminalregion on the glycosylation-free face and that dsRNA binds to only theglycosylation-free face of TLR3, such that two TLR3 monomers couldsandwich dsRNA in a complex of two TLR3 monomers and one dsRNA strand. Amodel involving an oligomeric complex of TLR3 formed by multiple dimersof TLR3 binding to longer dsRNA strands has also been proposed (see e.g.Bell et al. (2006) PNAS USA 103(23): 8792-8797). Ranjith-Kumar et al.(2007) J. Biol. Chem. 282(10): 7668-7677 propose that TLR3 can exist inan oligomerized stage in the absence of ligand, and that dsRNA bindingcan cause rearrangement in the dimer leading to lateral sliding of themonomers toward each other and adjust to accommodate the dsRNA, andwhere the resulting conformation change can stimulate the interaction ofthe TIR domain to induce signaling. One explanation could therefore bethat the anti-TLR3 antibodies 31C3, 29H3, 23C8, 28F11 and 34A3 bindTLR3, e.g. on glycosylation-free face, and possibly in an epitope havinga negative charge at neutral pH which is therefore not in the region ofdsRNA binding to TLR3. The antibodies therefore would not preventinteraction of dsRNA with TLR3, but could inhibit signaling, e.g. bypreventing a TLR3 dimer or oligomer that has bound dsRNA fromrearranging or adopting the configuration required to bring aboutinteraction of the TIR domains, or by interfering with the properformation of TLR3 dimers and/or a consequential TLR3-dsRNA ternarycomplex.

Example 5 Reporter Assay

Antibodies were tested for inhibition of TLR3 signaling in a luciferasebased reporter gene activity (293T-TLR3-ISRE). Engagement of TLR3receptor using TLR3-agonists such as poly (I:C) has been reported toactivate the type-IFN pathway including the promoter ISRE (Wietek et al.J. Biol. Chem., 278(51), p50923, 2003). Briefly, dsRNA TLR3 agonistswere used to induce TLR3 signaling in the reporter assay in the presenceof anti-TLR3 antibody 31C3, and TLR3 signaling was assessed. Theresults, shown in FIG. 9, show that anti-TL3 antibody 31C3 stronglyinhibited TLR3 signaling in a dose dependent fashion, compared to acontrol anti-TLR3 antibody previously determined to have no effect onTLR3 signaling.

In a further set of experiments, 293-huTLR3 cells were incubated withdifferent concentrations of the anti-TLR3 mAbs for 24 hours (see FIG.10A; mAb concentration is represented in axis in μg/ml on a logarithmicscale) followed by the addition to the medium of the TLR3 agonist polyAUat a concentration of 300 μg/ml. Luciferase expression was measuredafter 24 h based. The results are represented in FIG. 10A, and the IC50are represented in the Table 2 below indicating that the antibodiesaccording to the invention have excellent inhibition properties.

TABLE 2 IC50 (μg/ml) 31C3 2.25 23C8 1.83 28F11 5.56

In another set of experiments, the same inhibition test was carried outwith 100 μg/ml of polyAU and test antibodies 31C3, 23E8 and 34A3. IC50values were calculated and all of the antibodies had an IC50 below 5μg/ml. Furthermore, 34A3 exhibited an enhanced inhibition effect at lowconcentrations (FIG. 10B). FIGS. 10A and 10B show the inhibitionproperties of increasing doses of the antibodies according to theinvention, the inhibition of the TLR3 signaling is dose dependant. Thisassay confirms the excellent inhibition properties of the antibodiesaccording to the invention.

In a further test, the inhibition properties of the antibodies accordingto the invention were assessed in an inflammatory situation, here in thepresence of IFNα. Briefly, three different conditions were tested. Incondition 1 (FIG. 11A), IP-10 secretion was assessed without anypretreatment; in condition 2 (FIG. 11B), IP-10 secretion was assessedafter a 24 hours pre-treatment with IFNα (1000 UI/ml); in condition 3(FIG. 11C), IP-10 secretion was assessed after a 24 hours pre-treatmentwith polyAU (300 μg/ml), then a dose range of dsRNA (polyIC for FIGS.11A and 11B, polyAU for FIG. 11C) and the anti-TLR3 antibody, either31C3 or 23C8, at a concentration of 50 μg/ml, were added, IP-10secretion was quantified (in μg/ml) after 24 hours, using ELISA.

The results, represented in FIGS. 11A-11C, highlight that the antibodiesaccording to the invention remain good inhibitors of TLR3 even in thepresence of stimulatory molecules, either dsRNA or inflammatorycytokines. Such conditions mimic pre-established inflammatory diseases.

Example 6 FACS Staining

Briefly, 293T or 293T-ISRE/TLR3 cell lines are collected, the medium waswashed, and cells were fixed and permeabilized using an IntraPrep™permeabilization reagent from Beckman Coulter, following themanufacturer instruction. Permeabilized cells were then incubated at RTfor 20 min with 25 μg/mL of 31C3 antibody, further revealed by a goatanti-mouse antibody labeled with FITC. The graphs are represented inFIG. 13. Left panel represents the negative control on HEK293T cellline. Right panel represents the staining obtained on HEK293T-TLR3cells. Cells incubated with control isotype antibody, instead of 31C3anti-TLR3 antibody were unstained.

The staining obtained using antibody 31C3 in permeabilized cellsindicates that the antibodies specifically recognizes and bindsintracellular human TLR3 in cells.

Example 7 Kinetic Studies

Another set of assays were performed to determine the kinetics ofinhibition of the antibodies according to the invention. Briefly, MdDCwere incubated with an anti TLR3 antibody (31C3 or 23C8, at aconcentration of 50 μg/ml) and a dose range of polyAU in various timesettings. The medium was then incubated for 24 hours and IP-10 secretionwas measured by ELISA. FIG. 12A represents the IP-10 secretion in ng/ml(depending on the polyAU doses) for the 31C3 antibody; FIG. 12Brepresents the results for the 23C8 antibody.

Pre-stim: The anti-TLR3 antibody was incubated 1 h30 prior to dsRNAaddition.

Co-stim: The anti-TLR3 antibody and the dsRNA were added simultaneously.

Post-stim: The dsRNA was incubated 1 h30 prior to anti-TLR3 antibodyaddition

These results underline that the antibodies according to the inventionare efficient for TLR3 inhibition irrespective of the binding state ofthe dsRNA to the TLR3 protein. The antibodies do not compete with thebinding site of dsRNA but still are able to inhibit TLR3 signaling, evenwhen dsRNA is already bound to the TLR3 protein.

Example 8 TLR3 Internalization Assays

Briefly, either no antibody or 50 μg/mL of the anti-TLR3 antibody 31C3was incubated with live 293T-ISRE/TLR3 cell lines, for 2 h or 24 h at37° C. Cells were then washed, fixed and permeabilized using IntraPreppermeabilization reagent from Beckman Coulter. Presence of TLR3-boundanti-TLR3 31C3 Ab is revealed with a goat anti-mouse Ab, labelled withAPC. Alternatively, permeabilized cells were incubated with eithercontrol isotype or with a TLR3 specific mAb efficient in FACS,noncompeting with 31C3 Ab for TLR3 binding, both labelled with biotin,and further revealed through cell incubation with fluorescentstreptavidin derivative. The graphs are represented in FIGS. 20A and20B. FIG. 20A represents the negative control, representing fluorescenceintensity of the 293T-ISRE/TLR3 cells in the absence of an antibodylinking TLR3 proteins. FIGS. 20C and 20D represent the fluorescenceinduced by the binding to TLR3 protein of internalized 31C3 antibody,after 24 h or 2 h incubation, respectively. FIG. 20B indicates thesteady state level of TLR3 expression in 293T-ISRE/TLR3 cell lines,without pre-incubation with 31C3 antibody. FIGS. 20D and 20E, showing asimilar fluorescence than FIG. 20B confirm that the binding of TLR3 byantibody 31C3 does not down-modulate the expression of TLR3 on293T-ISRE/TLR3 cell lines.

Those results show that the antibodies according to the invention arerapidly and efficiently internalized, moreover without any hTLR3down-modulation. These results underline the efficiency of the bindingof the antibodies according to the invention. Furthermore, this rapidinternalization provides evidence that the antibodies are promisingcandidates for therapy. Such antibodies are also promising candidatesfor coupling with a toxic agent, thereby allowing a specific targetingof TLR3 expressing cells.

All headings and sub-headings are used herein for convenience only andshould not be construed as limiting the invention in any way. Anycombination of the above-described elements in all possible variationsthereof is encompassed by the invention unless otherwise indicatedherein or otherwise clearly contradicted by context. Recitation ofranges of values herein are merely intended to serve as a shorthandmethod of referring individually to each separate value falling withinthe range, unless otherwise indicated herein, and each separate value isincorporated into the specification as if it were individually recitedherein. Unless otherwise stated, all exact values provided herein arerepresentative of corresponding approximate values (e. g., all exactexemplary values provided with respect to a particular factor ormeasurement can be considered to also provide a correspondingapproximate measurement, modified by “about,” where appropriate).

All methods described herein can be performed in any suitable orderunless otherwise indicated herein or otherwise clearly contradicted bycontext.

The use of any and all examples, or exemplary language (e.g., “such as”)provided herein is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise indicated. No language in the specification should beconstrued as indicating any element is essential to the practice of theinvention unless as much is explicitly stated.

The citation and incorporation of patent documents herein is done forconvenience only and does not reflect any view of the validity,patentability and/or enforceability of such patent documents, Thedescription herein of any aspect or embodiment of the invention usingterms such as reference to an element or elements is intended to providesupport for a similar aspect or embodiment of the invention that“consists of,” “consists essentially of” or “substantially comprises”that particular element or elements, unless otherwise stated or clearlycontradicted by context (e. g., a composition described herein ascomprising a particular element should be understood as also describinga composition consisting of that element, unless otherwise stated orclearly contradicted by context).

This invention includes all modifications and equivalents of the subjectmatter recited in the aspects or claims presented herein to the maximumextent permitted by applicable law.

All publications and patent applications cited in this specification areherein incorporated by reference in their entireties as if eachindividual publication or patent application were specifically andindividually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to one of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

What is claimed is:
 1. A method for treating or preventing a diseaseselected from the group consisting of autoimmunity, inflammation,allergy, asthma, infection, cirrhosis and sepsis, said method comprisingadministering to a patient in need thereof a therapeutically effectiveamount of an antibody that specifically binds a human Toll-like receptor3 (TLR3) polypeptide, wherein said antibody inhibits signaling by saidhuman TLR3 polypeptide without blocking binding of a double-strandedribonucleic acid (dsRNA) TLR3 ligand to said human TLR3 polypeptide. 2.A method for treating or preventing a disease selected from the groupconsisting of autoimmunity, inflammation, allergy, asthma, infection,cirrhosis and sepsis, said method comprising administering to a patientin need thereof a therapeutically effective amount of an antibody thatspecifically binds a human Toll-like receptor 3 (TLR3) polypeptide,wherein said antibody specifically binds to said human TLR3 polypeptideunder acidic conditions with a sub-nanomolar K_(D) for its bivalentbinding affinity.
 3. A method for treating or preventing a diseaseselected from the group consisting of autoimmunity, inflammation,allergy, asthma, infection, cirrhosis and sepsis, said method comprisingadministering to a patient in need thereof a therapeutically effectiveamount of an antibody that specifically binds a human Toll-like receptor3 (TLR3) polypeptide, wherein said antibody has respectively a VH and VLregion of SEQ ID NO: 2 and 3 (31C3).
 4. The method of claim 1, furthercomprising the step of administering to the patient an appropriateadditional therapeutic agent selected from the group consisting of animmunomodulatory agent, a corticosteroid, an immunosuppressant, anantibiotic, an antiviral and an anti-inflammatory agent.
 5. The methodof claim 2, further comprising the step of administering to the patientan appropriate additional therapeutic agent selected from the groupconsisting of an immunomodulatory agent, a corticosteroid, animmunosuppressant, an antibiotic, an antiviral and an anti-inflammatoryagent.
 6. The method of claim 3, further comprising the step ofadministering to the patient an appropriate additional therapeutic agentselected from the group consisting of an immunomodulatory agent, acorticosteroid, an immunosuppressant, an antibiotic, an antiviral and ananti-inflammatory agent.
 7. A method for treating or preventing acancer, comprising administering to a patient in need thereof atherapeutically effective amount of an antibody that specifically bindsa human Toll-like receptor 3 (TLR3) polypeptide, wherein said antibodyinhibits signaling by said human TLR3 polypeptide without blockingbinding of a double-stranded ribonucleic acid (dsRNA) TLR3 ligand tosaid human TLR3 polypeptide, and wherein said antibody is conjugated orcovalently bound to a toxic moiety.
 8. A method for treating orpreventing a cancer, comprising administering to a patient in needthereof a therapeutically effective amount of an antibody thatspecifically binds a human Toll-like receptor 3 (TLR3) polypeptide,wherein said antibody specifically binds to said human TLR3 polypeptideunder acidic conditions with a sub-nanomolar K_(D) for its bivalentbinding affinity, and wherein said antibody is conjugated or covalentlybound to a toxic moiety.
 9. A method for treating or preventing acancer, comprising administering to a patient in need thereof atherapeutically effective amount of an antibody that specifically bindsa human Toll-like receptor 3 (TLR3) polypeptide, wherein said antibodyhas respectively a VH and VL region of SEQ ID NO: 2 and 3 (31C3), andwherein said antibody is conjugated or covalently bound to a toxicmoiety.
 10. The method of claim 7, wherein said antibody is capable ofbeing internalized by a TLR3-expressing cell.
 11. The method of claim 8,wherein said antibody is capable of being internalized by aTLR3-expressing cell.
 12. The method of claim 9, wherein said antibodyis capable of being internalized by a TLR3-expressing cell.
 13. A methodfor treating or preventing a disease wherein the inhibition of TLR3signaling pathway is desirable, said method comprising administering atherapeutically effective amount of an antibody to a patient in needthereof, wherein the antibody specifically binds a human Toll-likereceptor 3 (TLR3) polypeptide, and wherein said antibody is selectedfrom the group consisting of: (a) an antibody having respectively a VHand VL region of SEQ ID NO: 34 and 35 (34A3), (b) an antibody havingrespectively a VH and VL region of SEQ ID NO: 10 and 11 (29H3), (c) anantibody having respectively a VH and VL region of SEQ ID NO: 18 and 19(28F11), and (d) an antibody having respectively a VH and VL region ofSEQ ID NO: 26 and 27 (23C8).
 14. The method of claim 13, wherein saiddisease is selected from the group consisting of autoimmunity,inflammation, allergy, asthma, infection, cirrhosis, sepsis, diabetes,and viral infections.
 15. A method for treating or preventing a diseasewherein the inhibition of TLR3 signaling pathway is desirable, saidmethod comprising administering a therapeutically effective amount of anantibody to a patient in need thereof, wherein said antibody competesfor binding to a TLR3 polypeptide with any one or any combination ofmonoclonal antibodies 31C3, 29H3, 23C8, 28F11, and 34A3.
 16. The methodof claim 15, wherein said disease is selected from the group consistingof autoimmunity, inflammation, allergy, asthma, infection, cirrhosis,sepsis, diabetes, and viral infections.
 17. A method for treating orpreventing a disease wherein the inhibition of TLR3 signaling pathway isdesirable, said method comprising administering a therapeuticallyeffective amount of an antibody to a patient in need thereof, whereinsaid antibody is specific for an epitope in the human Toll-like receptor3 (TLR3) polypeptide that does not undergo substantial change inelectrostatic potential or undergoes less change than a region ofpositive electrostatic potential when neutral conditions are acidifiedsuch that the binding affinity of said antibody remains substantiallyunchanged.
 18. The method of claim 17, wherein said disease is selectedfrom the group consisting of autoimmunity, inflammation, allergy,asthma, infection, cirrhosis, sepsis, diabetes, and viral infections.19. A method for treating or preventing a disease wherein the inhibitionof TLR3 signaling pathway is desirable, said method comprisingadministering a therapeutically effective amount of an antibody to apatient in need thereof, wherein said antibody specifically binds to thesame human Toll-like receptor 3 (TLR3) epitope as any one or anycombination of monoclonal antibodies 31C3, 29H3, 23C8, 28F11 or 34A3.20. The method of claim 19, wherein said disease is selected from thegroup consisting of autoimmunity, inflammation, allergy, asthma,infection, cirrhosis, sepsis, diabetes, and viral infections.